Receptor/histidine kinase fusion constructs and uses thereof

ABSTRACT

The present disclosure provides compositions and methods for sensing a target substance of interest in the environment and inducing gene expression in response thereto, useful for detection of biological and chemical agents and environmental pollutants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Appl. Ser. No.62/701,396, filed Jul. 20, 2018, the entire disclosure of which isincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grantW911NF-09-1-0526 awarded by Army Research Office and grantN00014-07-1-0180 awarded by the Office of Naval Research. The governmenthas certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

A sequence listing contained in the file named “CSUV004US.txt” which is172 kilobytes (measured in MS-Windows®) and created on Jun. 18, 2019, isfiled electronically herewith and incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present disclosure relates to the field of molecular biology, morespecifically compositions and methods involved in signal transductionfrom outside of a cell to the nucleus and to systems for sensing atarget substance of interest in the environment and inducing geneexpression in response thereto, useful for detection of biological andchemical agents, pathogens and their products and environmentalpollutants especially with plants and plant sentinels.

BACKGROUND

Current detectors of biological and chemical agents and environmentalpollutants involve electronic and/or vacuum-like mechanisms to samplethe air or the environment. All current means to detect harmfulbiological or chemical agents and environmental pollutants are costlyand require continuous maintenance. The high and continuous costsignificantly limits the ability to detect biological and chemicalagents, pathogens and their products, as well as environmentalpollutants.

Therefore there is an increasing need for simple and robust detectorsfor harmful biological or chemical agents, pathogens and their products,and environmental pollutants.

SUMMARY OF THE INVENTION

The present disclosure provides a fusion protein comprising achemotactic receptor protein, or a receptor involved in quorum sensing,or a receptor from a receptor histidine kinase operably linked at theA/D position to a histidine kinase protein, wherein the fusion proteincomprises a kinase activation region. In certain embodiments thechemotactic receptor protein is Trg, Tar, Tap or Tsr. In otherembodiments the receptor involved in quorum sensing is the Xylella DSFreceptor RpfC or the LuxPQ receptor LuxP. In additional embodiments thehistidine kinase protein is PhoR or EnvZ, while in further embodimentsthe histidine kinase protein is a EnvZ/PhoR chimera. In some embodimentsthe kinase activation region of the fusion protein has been engineeredto restore inducible kinase activity or engineered to allow theinteraction of maltose-bound maltose binding protein with the receptorto functionally activate kinase activity.

In particular embodiments the histidine kinase protein is activated whenthe chemotactic receptor protein or the receptor involved in quorumsensing binds to a sensor protein bound to a target substance or atarget substance itself. Because of the diversity that nature providesfrom histidine kinases and with the ability afforded by computationaldesign of proteins such as demonstrated here, in certain embodiments thefusions can be made where the target substance is a chemical agent, aheavy metal, a poison, a pollutant, a toxin, an herbicide, a polycyclicaromatic hydrocarbon, a benzene, a toluene, a xylene, a halogenatedhydrocarbon, a steroid or other hormone, an explosive, or a degradationproduct of one of the foregoing compounds, most small molecules asenabled with computational design. In further embodiments the fusionprotein further comprises a plasma membrane targeting signal sequenceoperably linked to an N-terminus of the chemotactic receptor protein orreceptor involved in quorum sensing. Quorum sensing molecules are foundin pathogen and their related products as demonstrated here and expandedwith computational design abilities, hence our embodiments here and withcomputational abilities provide means to sense and respond to pathogens.

The present disclosure also provides a DNA construct comprising anucleic acid segment that encodes a fusion protein comprising achemotactic receptor protein, a receptor involved in quorum sensing, ora receptor from a receptor histidine kinase operably linked at the A/Dposition to a histidine kinase protein, wherein the fusion proteincomprises a kinase activation region. In some embodiments the nucleicacid segment is operably linked to a heterologous or homologouspromoter. In further embodiments the chemotactic receptor protein or thereceptor involved in quorum sensing are or can be computationallydesigned.

The present disclosure further provides a transgenic plant or a plantcell comprising a first DNA construct comprising a first plant operablepromoter operably linked to a nucleic acid segment encoding a sensorprotein, the protein comprising a secretory sequence for directing theprotein to the extracellular space of a plant cell and a binding regionspecific for a target substance of interest, wherein the proteinundergoes a conformational change when the target substance is bound, asecond DNA construct comprising a second plant operable promoteroperably linked to a nucleic acid segment encoding a protein thatcomprises the following domains: a plasma membrane targeting signalsequence, an extracellular domain for binding the sensor protein, atransmembrane domain and a histidine kinase domain for phosphorylating aprotein with nuclear shuttling or transcriptional activating functions,wherein the histidine kinase is activated when the sensor protein bindsto the extracellular domain, and a third DNA construct comprising athird plant operable promoter operably linked to a nucleic acid segmentencoding a detectable marker or a response gene, wherein the third plantoperative promoter is responsive to the transcriptional activatorprotein, and wherein the detectable marker is expressed when theexternal target substance of interest is bound to the sensor protein. Inparticular embodiments the extracellular domain for binding the sensorprotein comprises a chemotactic receptor protein, a receptor involved inquorum sensing, or a receptor from a receptor histidine kinase, and theextracellular domain or the transmembrane domain is operably linked atthe A/D position to a histidine kinase protein, wherein the second DNAconstruct encodes a fusion protein comprising a kinase activationregion.

In certain embodiments the extracellular domain, the transmembranedomain and/or the histidine kinase domain of the second DNA constructare derived from one or more bacterial genes, and the membrane targetingsignal sequence of the second DNA construct is derived from a plantgene. The derived bacterial genes can be subjected to refactoring (codonoptimization, removal of splice sites, post-translational regulatoryelements, etc.) as is typical for one skilled in the field. In someembodiments the detectable marker of the third DNA construct is achlorophyll degradation enzyme or a functional fragment thereof. Inother embodiments the plant loses detectable green color when thedetectable marker is expressed. In yet other embodiments the chlorophylldegradation enzyme is selected from the group consisting of redchlorophyll catabolite reductase (RCCR), pheophorbide a oxygenase (PaO),and chlorophyllase. In still other embodiments the third DNA constructcomprises a plant operable promoter responsive to a transcriptionactivator protein operably linked to a nucleic acid sequence encoding aninterfering RNA molecule specific for a chlorophyll biosynthesis codingsequence. In further embodiments the chlorophyll biosynthesis codingsequence encodes chlorophyll synthetase, protochlorophyllideoxidoreductase (POR) or GUN4. In other embodiments the response orreadout is non-visible but detectable by various systems includingwebcams and high altitude platforms.

In particular embodiments the secretory sequence is from pollenextension-like protein (PEX). In certain embodiments the membranetargeting signal sequence is from FLS2. In further embodiments thehistidine kinase domain comprises segments derived from a non-plantorganism or segments derived from a non-plant organism and a plant. Inadditional embodiments the plant operable promoter comprises a PhoBbinding sequence.

In further embodiments the transgenic plant or plant cell furthercomprises a fourth DNA construct comprising a nucleic acid encoding achlorophyll degradation enzyme or a functional fragment thereof operablylinked to a plant operable promoter responsive to the transcriptionactivator protein, and wherein the promoter is not in nature associatedwith the sequence encoding a chlorophyll degradation enzyme. In yetfurther embodiments the transgenic plant or plant cell further comprisesa fourth DNA construct comprising a plant operable promoter operablylinked to a nucleic acid sequence encoding a plant operabletranscriptional activator, wherein the transcriptional activator isactivated when phosphorylated by a histidine kinase.

In certain embodiments the detectable marker is a functional RNA. Insome embodiments the functional RNA is an interfering RNA molecule. Inother embodiments the functional RNA inhibits expression of achlorophyll biosynthesis coding sequence. In particular embodiments thechlorophyll biosynthesis coding sequence encodes chlorophyll synthetase,protochlorophyllide oxidoreductase (POR) or GUN4. In additionalembodiments the detectable marker is a chlorophyll degradation enzyme.In certain embodiments the chlorophyll degradation enzyme is redchlorophyll catabolite reductase (RCCR), pheophorbide a oxygenase (PaO),or chlorophyllase. In yet other embodiments the detectable marker is aβ-glucuronidase, a β-galactosidase or a green or yellow fluorescentprotein.

In some embodiments the transcription activator protein comprises aresponse regulator domain. In certain embodiments the response regulatordomain is derived from PhoB. In particular embodiments the transcriptionactivator protein is a PhoB:VP64 translational fusion protein. Infurther embodiments the detectable marker is a functional RNA thatinhibits expression of a chlorophyll biosynthesis coding sequence. Instill further embodiments the plant loses green color due to inhibitionof chlorophyll biosynthesis and enhanced breakdown of chlorophyll uponinduction of a gene encoding a chlorophyll degradation enzyme. In otherembodiments the enhanced breakdown of chlorophyll is achieved byexpressing at least one enzyme selected from the group consisting of redchlorophyll catabolite reductase (RCCR), pheide a oxygenase (PaO), andchlorophyllase. In yet other embodiments the inhibition of chlorophyllbiosynthesis is achieved by inhibiting expression of at least one enzymeselected from the group consisting of protochlorophyllide oxidoreductase(POR), chlorophyll synthetase and GUN4. In certain embodiments theinhibition of POR is achieved by producing an interfering RNA moleculethat contains a sequence derived from the coding sequence of POR. Infurther embodiments the plant loses green color by inhibiting POR andstimulating RCCR and chlorophyllase. In other embodiments the responseor readout is non-visible but detectable by various systems includingwebcams and high altitude platforms.

The present disclosure additionally provides a plant cell comprising afirst DNA construct comprising a first promoter functional in a plantoperably linked to a recombinant nucleic acid encoding a firstrepressor, a second DNA construct comprising a second promoterfunctional in a plant operably linked to a nucleic acid encoding adetectable marker or response gene encoding a fusion protein comprisinga chemotactic receptor protein, a receptor involved in quorum sensing,or a receptor from a receptor histidine kinase operably linked at theA/D position to a histidine kinase protein, wherein the fusion proteincomprises a kinase activation region, wherein the second promoter isrepressible by a second repressor, and a third DNA construct comprisinga third promoter functional in a plant operably linked to a nucleic acidencoding the first repressor, wherein the third promoter is constitutiveand repressible by the second repressor, wherein the first repressor orsecond repressor comprise at least one EAR1 or EAR 2 repressor domain,or a transgenic plant comprising the plant cell. In further embodimentsthe plant cell comprises a fourth DNA construct comprising a fourthpromoter operable in a plant operably linked to a nucleic acid encodingthe second repressor, wherein the fourth promoter is constitutive andrepressible.

In certain embodiments the fourth promoter is repressible by the firstrepressor. In some embodiments repression of the fourth promoter by thefirst repressor reduces expression of the second repressor. In otherembodiments reduced expression of the second repressor increasesexpression of the detectable marker or response gene. In yet otherembodiments the fourth promoter is a recombinant polynucleotidecomprising nucleic acid sequence from a non-plant organism. In stillother embodiments the nucleic acid encoding the first repressor is arecombinant polynucleotide. In particular embodiments the nucleic acidencoding the first repressor comprises nucleic acid sequences encodingat least one GAL4 DNA binding domain. In further embodiments the nucleicacid encoding the second repressor is a recombinant polynucleotide. Inyet further embodiments the first promoter is a recombinantpolynucleotide. In still further embodiments the first promoter isinduced by a transcription activator protein activated by an externalsignal.

In additional embodiments the transcription activator protein is afusion protein encoded by a polynucleotide sequence derived from anon-plant organism. In other embodiments the polynucleotide sequenceencoding the fusion protein comprises at least one nucleic acid sequenceencoding a PhoB binding domain. In some embodiments the fusion proteinis encoded by a polynucleotide sequence comprising a nucleic acidsequence encoding a polypeptide sequence of VP64. In further embodimentsthe plant cell comprises a fifth DNA construct comprising a plantoperable promoter operably linked to a nucleic acid sequence encoding asensor protein that recognizes the external signal. In certainembodiments the second promoter is a recombinant polynucleotide.

The present disclosure also provides a method for detecting an externalsubstance of interest, the method comprising exposing a transgenic plantor a plant cell comprising a first DNA construct comprising a firstplant operable promoter operably linked to a nucleic acid segmentencoding a sensor protein, the protein comprising a secretory sequencefor directing the protein to the extracellular space of a plant cell anda binding region specific for a target substance of interest, whereinthe protein undergoes a conformational change when the target substanceis bound, a second DNA construct comprising a second plant operablepromoter operably linked to a nucleic acid segment encoding a proteinthat comprises the following domains: a plasma membrane targeting signalsequence, an extracellular domain for binding the sensor protein, atransmembrane domain and a histidine kinase domain for phosphorylating aprotein with nuclear shuttling and/or transcriptional activatingfunctions, wherein the histidine kinase is activated when the sensorprotein binds to the extracellular domain, and a third DNA constructcomprising a third plant operable promoter operably linked to a nucleicacid segment encoding a detectable marker or a response gene, whereinthe third plant operative promoter is responsive to the transcriptionalactivator protein, and wherein the detectable marker is expressed whenthe external target substance of interest is bound to the sensorprotein, wherein the extracellular domain for binding the sensor proteincomprises a chemotactic receptor protein, a receptor involved in quorumsensing, or a receptor from a receptor histidine kinase, and theextracellular domain or the transmembrane domain is operably linked atthe A/D position to a histidine kinase protein, wherein the second DNAconstruct encodes a fusion protein comprising a kinase activation regionto an external substance of interest, and detecting a change resultingfrom expression of the detectable marker.

In certain embodiments the detectable marker is a functional RNA. Insome embodiments the functional RNA is an interfering RNA molecule. Inother embodiments the functional RNA inhibits expression of achlorophyll biosynthesis coding sequence. In yet other embodiments thechlorophyll biosynthesis coding sequence encodes chlorophyll synthetase,protochlorophyllide oxidoreductase (POR) or GUN4. In still otherembodiments the detectable marker is a chlorophyll degradation enzyme.In further embodiments the chlorophyll degradation enzyme is redchlorophyll catabolite reductase (RCCR), pheophorbide a oxygenase (PaO),or chlorophyllase. In still further embodiments the detectable marker isa β-glucuronidase, a β-galactosidase or a green or yellow fluorescentprotein. In other embodiments the response or readout is non-visible butdetectable by various systems including webcams and high altitudeplatforms. In additional embodiments the transcription activator proteinis a PhoB protein or is derived from a PhoB protein. In some embodimentsthe transcription activator protein is a PhoB:VP64 translational fusionprotein.

In certain embodiments the change is degreening of the transgenic plant.In some embodiments the degreening of the transgenic plant is detectedvisually or by detecting properties selected from the group consistingof chlorophyll fluorescence, photosynthetic properties and propertiesrelated to reactive oxygen species and their damage. In otherembodiments the degreening is detected by imaging selected from thegroup consisting of hyper-spectral imaging, infra-red imaging,near-infra-red imaging and multi-spectral imaging. In furtherembodiments the transgenic plant regreens after removal of the externalsubstance of interest. In other embodiments the response or readout isnon-visible but detectable by various systems including webcams and highaltitude platforms. In another embodiment the external signal isdetectable after a single exposure of the transgenic plant or plant cellto the external signal.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent disclosure, reference is now made to the detailed description ofthe disclosure along with the accompanying figures. The drawingsdescribed herein are for illustration purposes only and are not intendedto limit the scope of the present teachings in any way.

FIG. 1A, FIG. 1B and FIG. 1C: Diagrams of two component systems (TCS).FIG. 1A. “Simple” bacterial TCS—Extracellular input is provided by atransmembrane histidine kinase (HK). In the HK molecule a histidineresidue in the dimerization and histidine phosphorylation (DHP) domainauto-phosphorylates in response to ligand binding. The high energyphosphoryl group is then transferred to a cytoplasmically localizedresponse regulator (RR). FIG. 1B. Hybrid HK signaling found in plantsand bacteria—These systems have more complex HKs and signalingcomponents. In response to a ligand binding on the HK, a histidineresidue in the DHP domain auto-phosphorylates. The high energyphosphoryl group is then transferred internally to an aspartate in thereceiver domain, and subsequently to a histidine in a cytoplasmic Hptprotein which then phosphorylates an RR on its aspartate residue. FIG.1C. Bacterial chemotactic TCS—A chemotactic receptor (e.g., Trg) binds aPBP-ligand complex or directly binds a ligand. This binding eventinitiates a signal that is transmitted to a cytoplasm-localizedhistidine kinase CheA. CheW acts as an adaptor protein for thechemotactic receptor/CheA complex. CheA then phosphorylates RRs that donot activate transcription.

FIG. 2 . Synthetic Signaling System Used In Detector Plants. In theapoplast, computationally re-designed PBPs bind TNT. The PBP-TNT complexinteracts with a membrane-localized Trg-PhoR (DHP8) fusion, causing itto auto-phosphorylate and transfer the high energy phosphate toPhoB-VP64. PhoB-VP64 translocates to the nucleus and activates thePlantPho promoter.

FIG. 3A, FIG. 3B and FIG. 3C. Interaction between the two α-helices inthe tropomyosin coiled-coil. Each α-helix is shown with seven residues(a-g) in two turns. FIG. 3A. End-on view looking from N terminus. Theinterface between the α-helices derives primarily from hydrophobicresidues in core positions a and d, although there are also some saltbridges formed between residues e and g. FIG. 3B. The core interfaceviewed parallel to the coiled-coil axis shows how residues from onechain occupy the spaces between the corresponding residues from thesecond chain to give “knobs in holes” packing. FIG. 3C. Representationof a coiled coil structure.

FIG. 4 . Predicted HK coiled coil element in Trz and PhoR. Note that themethionine used in the HAMP fusion between Trg and EnvZ (underlined) isadjacent to the start of coiled coil heptad number 3.

FIG. 5 . Predicted coiled coil and fusions junctions from coiled coiltest constructs. Proteins used in the fusions (EnvZ—SEQ ID NO:50;Trg—SEQ ID NO:51; Trz—SEQ ID NO:52; PhoR—SEQ ID NO:53) highlighted inblue. Sequence junctions from the 4 coiled coil test constructs(Trg″CC″Pho—SEQ ID NO:54; TrzEnvZCC—SEQ ID NO:55; TrzEnvZcc2&3—SEQ IDNO:56; TrgPhoRcc3—SEQ ID NO:57) are shown along with the signalingphenotype. The autophosphorylated Histidine from the DHP domain is shownin bold. The position of the HAMP helices, if found in the protein, areshown above the alignments.

FIG. 6 . pACYC177 based fusion testing plasmid.

FIG. 7 . Refining the TrzPhoR functional fusion point. Proteins used inthe fusions are Trg (SEQ ID NO:51); EnvZ (SEQ ID NO:50); Trg″cc1″PhoR(SEQ ID NO:54); and PhoR (SEQ ID NO:53). TrzHAMP+VK (SEQ ID NO:60)recapitulates the original coiled coil based TrzPhoR fusion (SEQ IDNO:58). Residues efg of the Trg HAMP domain (QHS vs AAG in EnvZ) canfunctionally substitute for the EnvZ efg residues from the originalTrzHAMP fusion as seen in TrgHAMP+V (SEQ ID NO:61) and TrzHAMP+V PhoR(SEQ ID NO:59).

FIG. 8 . Coiled coil D position mutants TrzHAMP+M (SEQ ID NO:62),TrzHAMP+V (SEQ ID NO:63), TrgHAMP+V (SEQ ID NO:64), TrgHAMP+G (SEQ IDNO:65), TrgHAMP+A (SEQ ID NO:66), TrgHAMP+L (SEQ ID NO:67), TrgHAMP+I(SEQ ID NO:68), TrgHAMP+E (SEQ ID NO:69), and TrgHAMP+T (SEQ ID NO:70).Proteins used in the fusions are Trz (SEQ ID NO:52); Trg (SEQ ID NO:51);and PhoR (SEQ ID NO:53). TrgHAMP+G and TrgHAMP+A tested the effect ofputting a smaller hydrophobic D position residue in place of the Valine.TrgHAMP+L,I,E or T tested the effect of placing a naturally occurringA/D residue from the 10 HKs found in E. coli that have the HAMP positionA/D position HK Coiled coil architecture.

FIG. 9A and FIG. 9B. Imaging of a luciferase gene reporter in transgenicArabidopsis with the signaling circuit Ribose Binding Protein(RBP)→TrzPhoR→PhoBVP64 activating a plant pho promoter: luciferasereporter gene FIG. 9A. Four transgenic Arabidopsis lines at 0 hours (noexposure to ribose) top panel and 24 hours after exposure to ribosebottom panel. FIG. 9B. Quantification of luciferase activity from FIG.9A.

FIG. 10 . β-galactosidase assay showing diffusible signaling factorsignaling via the RpfCPhoR fusion. Yellow=control, Blue=DSF.

FIG. 11A and FIG. 11B. FIG. 11A. Paired detached leaf assay on a splitplate with luciferase reporter readout, 24 hour exposure to DSF. Twoleaves from 7 independent RpfCTrzPhoR transgenic lines were detached andplaced on media without (left side) and with (right side) DSF extractedfrom Xylella. The signaling circuit is DSF→RpfCTrzPhoR→PhoBVP64activating a plant pho promoter. FIG. 11B. Quantification of luciferaseactivity. Mean activity of all 7 leaves.

FIG. 12A and FIG. 12B. FIG. 12A. Split plate assay showing Mg²⁺induction of PhoQTrzPhoR. Left=background signaling from 2 mM MgSO₄present in media, Right=signaling increase seen with addition of 10 mMMgCl₂. FIG. 12B. β-galactosidase activity of PhoQTrzPhoR.Yellow=background signaling from Mg²⁺ in media, Blue=signaling withadditional 10 mM Mg²⁺.

FIG. 13A and FIG. 13B. β-galactosidase activity of TrzChim3 and theTrzChim3 ADD→EGA mutant. FIG. 13A. β-galactosidase activity of TrzChim3.Yellow=control, Blue=ribose. FIG. 13B. β-galactosidase activity of theTrzChim3 ADD→EGA mutant. Yellow=control, Blue=ribose.

FIG. 14 . Alignment between EnvZ (SEQ ID NO:71) and PhoR (SEQ ID NO:72)indicating the ADD residues (275-277) of EnvZ and the corresponding EGAresidues of PhoR in bold underline.

FIG. 15 . Split plate assay showing ribose induction of the TrzChimvariants 8 and 10. Left side=control, right side=ribose.

FIG. 16 . β-galactosidase activity of TrzChim3, TrzChim3-8 andTrzChim3-10. Yellow=control, Blue=ribose.

FIG. 17A and FIG. 17B. Normalized GFP reporter gene fluorescence showingmaltose induction of the TarHK fusion variants TazPhoR (FIG. 17A) andTAC (FIG. 17B).

FIG. 18 . Data showing function of computationally designed protein tofentanyl with histidine kinase fusions and synthetic signal transductionsystem. Plants without (Control) and with exposure to the ligand(fentanyl) were evaluated.

FIG. 19 . Means to engineer plant sensing and response systems. Leftportion shows means to produce a rapid response in a plant. Acomputationally designed protein is located in the apoplast. When thisprotein binds a ligand, there is a conformational change that produces aresponse or readout. Right portion shows two means to use the synthetichistidine kinase systems. In one case (center) the computationallydesigned protein, partially or entirely, functions as a periplasmicprotein (e.g., MBP). When the ligand binds the protein, there is highaffinity for the external domain of the synthetic or computationallydesigned histidine kinase (chemotactic receptor/HK protein). Aconformational change takes places that activates that transmembranemolecule, specifically, the internal kinase domain. This initiates aphosphoryl relay with a modified response regulator (e.g., engineeredPhoB, OmpR) or computational designs of these, that translocates in theplant nucleus, binds a receptive promoter and activates atranscriptional response. The response can produce a direct readout, orthe response can be regulated or tuned as shown schematically in FIG. 20and demonstrated in FIG. 18 . The second means to initiate a response isshown on the far right. A transmembrane fusion is made with a pathogenreceptor-histidine kinase as shown with RpfC. The pathogen or pathogenfactor activates the receptor-HK fusion protein and initiates a responseas shown for the MBP protein (center).

FIG. 20 . Genetic means to regulate or tune a plant sensing signal asused in FIG. 18 . Once the transcriptional signal is received by thereceptive promoter, in this case the PlantPho promoter, a quantitativelytuned response cascade is initiated. Transcription from the PlantPhopromoter activates a second transcription factor, Tal-Gal4, thatactivates the pUAS promoter driving GAL4-VP64. Expression of GAL4-VP64feeds-back and produces more of its own expression and feeds-forward toinitiate expression of any response, readout or reporter gene.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1—DNA coding sequence of TrzPhoR coiled coil based fusion ofTrz (Trg chemotactic receptor/EnvZ histidine kinase fusion) to PhoRhistidine kinase Escherichia coli sequence.

SEQ ID NO:2—Protein sequence of TrzPhoR coiled coil based fusion of Trz(Trg chemotactic receptor/EnvZ histidine kinase fusion) to PhoRhistidine kinase Escherichia coli sequence.

SEQ ID NO:3—DNA coding sequence of TrzPhoR: Trz HAMP domain+Valine A/Dfusion to PhoR histidine kinase Escherichia coli sequence.

SEQ ID NO:4—Protein sequence of TrzPhoR: Trz HAMP domain+Valine A/Dfusion (SEQ ID NO:48) to PhoR histidine kinase Escherichia coli sequence(SEQ ID NO:49).

SEQ ID NO:5—DNA coding sequence of RpfCPhoR Fusion of Xylella fastidiosaRpfC quorum sensing receptor to PhoR HK at A/D valine position,Arabidopsis codon optimized sequence.

SEQ ID NO:6—Protein sequence of RpfCPhoR Fusion of Xylella fastidiosaRpfC quorum sensing receptor to PhoR HK at A/D valine position.Arabidopsis codon optimized sequence.

SEQ ID NO:7—DNA coding sequence of TrzChim3: Trz with partialsubstitution of PhoR DHP domain Escherichia coli sequence.

SEQ ID NO:8—Protein sequence of TrzChim3: Trz with partial substitutionof PhoR DHP domain Escherichia coli sequence.

SEQ ID NO:9—DNA coding sequence of TrzChim3 ADD to EGA variantEscherichia coli sequence.

SEQ ID NO:10—Protein sequence of TrzChim3 ADD to EGA variant Escherichiacoli sequence.

SEQ ID NO:11—DNA coding sequence of TrzChim3-8 Directed evolutionvariant of TrzChim3 with restored on/off functionality Escherichia colisequence.

SEQ ID NO:12—Protein sequence of TrzChim3-8 Directed evolution variantof TrzChim3 with restored on/off functionality Escherichia colisequence.

SEQ ID NO:13—DNA coding sequence of TrzChim3-10 Directed evolutionvariant of TrzChim3 with restored on/off functionality Escherichia colisequence.

SEQ ID NO:14—Protein sequence of TrzChim3-10 Directed evolution variantof TrzChim3 with restored on/off functionality Escherichia colisequence.

SEQ ID NO:15—DNA coding sequence of TazPhoR: TrzPhoR fusion with the Tarchemotactic receptor replacing the Trg chemotactic receptor Escherichiacoli sequence.

SEQ ID NO:16—Protein sequence of TazPhoR: TrzPhoR fusion with the Tarchemotactic receptor replacing the Trg chemotactic receptor Escherichiacoli sequence.

SEQ ID NO:17—DNA coding sequence of TazPhoR 61: Maltose inducibledirected evolution variant of TazPhoR Escherichia coli sequence.

SEQ ID NO:18—Protein sequence of TazPhoR 61: Maltose inducible directedevolution variant of TazPhoR Escherichia coli sequence.

SEQ ID NO:19—DNA coding sequence of TazPhoR 86: Maltose inducibledirected evolution variant of TazPhoR Escherichia coli sequence.

SEQ ID NO:20—Protein sequence of TazPhoR 86: Maltose inducible directedevolution variant of TazPhoR Escherichia coli sequence.

SEQ ID NO:21—DNA coding sequence of Tac8: TrzChim3-8 with thechemotactic receptor Tar replacing the chemotactic receptor TrgEscherichia coli sequence.

SEQ ID NO:22—Protein sequence of Tac8: TrzChim3-8 with the chemotacticreceptor Tar replacing the chemotactic receptor Trg Escherichia colisequence.

SEQ ID NO:23—DNA coding sequence of Tac40: Maltose inducible directedevolution variant of Tac8.

SEQ ID NO:24—Protein sequence of Tac40: Maltose inducible directedevolution variant of Tac8.

SEQ ID NO:25—DNA coding sequence of PhoR Phosphate regulon sensorHistidine Kinase from Escherichia coli.

SEQ ID NO:26—Protein sequence of PhoR Phosphate regulon sensor HistidineKinase from Escherichia coli.

SEQ ID NO:27—DNA coding sequence of Trg Ribose responsive chemotacticreceptor that interacts with ribose binding protein (RBP) fromEscherichia coli.

SEQ ID NO:28—Protein sequence of Trg Ribose responsive chemotacticreceptor that interacts with ribose binding protein (RBP) fromEscherichia coli.

SEQ ID NO:29—DNA coding sequence of Tar Aspartate and Maltose responsivechemotactic receptor that interacts with maltose binding protein fromEscherichia coli.

SEQ ID NO:30—Protein sequence of Tar Aspartate and Maltose responsivechemotactic receptor that interacts with maltose binding protein fromEscherichia coli.

SEQ ID NO:31—DNA coding sequence of EnvZ Histidine kinase involved inosmotic sensing from Escherichia coli.

SEQ ID NO:32—Protein sequence of EnvZ Histidine kinase involved inosmotic sensing from Escherichia coli.

SEQ ID NO:33—DNA coding sequence of RpfC Quorum sensing histidine kinasethat senses diffusible signaling factor DSF from Xylella fastidiosa.

SEQ ID NO:34—Protein sequence of RpfC Quorum sensing histidine kinasethat senses diffusible signaling factor DSF from Xylella fastidiosa.

SEQ ID NO:35—DNA coding sequence of Trz-Trg chemotactic receptor fusedto EnvZ histidine kinase at conserved HAMP domain: Trg nt 1-801_EnvZ nt802-end.

SEQ ID NO:36—Protein sequence of Trz-Trg chemotactic receptor fused toEnvZ histidine kinase at conserved HAMP domain: Trg aa 1-275_EnvZ aa276-end.

SEQ ID NO:37—DNA coding sequence of Taz-Tar chemotactic receptor fusedto EnvZ histidine kinase at conserved HAMP domain: Tar nt 1-778_EnvZ nt779-end.

SEQ ID NO:38—Protein sequence of Taz-Tar chemotactic receptor fused toEnvZ histidine kinase at conserved HAMP domain: Tar aa 1-262_EnvZ aa263-end.

SEQ ID NO:39—DNA coding sequence of PhoQTrzPhoR: Mg²⁺ responsive PhoQreceptor fused to TzyPhoR at the D position. Escherichia coli sequence.

SEQ ID NO:40—Protein sequence of PhoQTrzPhoR: Mg²⁺ responsive PhoQreceptor fused to TzyPhoR at the D position. Escherichia coli sequence.

SEQ ID NO:41—DNA coding sequence of LuxQTrzPhoR: D position fusion ofreceptor histidine kinase LuxA which quorum senses AI-2 and TrzPhoR LuxQcodon optimized for Escherichia coli. TrzPhoR Escherichia coli sequence.

SEQ ID NO:42—Protein sequence of LuxQTrzPhoR: D position fusion ofreceptor histidine kinase LuxA which quorum senses AI-2 and TrzPhoR LuxQcodon optimized for Escherichia coli. TrzPhoR Escherichia coli sequence.

SEQ ID NO:43—PhoQ DNA coding sequence from Escherichia coli.

SEQ ID NO:44—PhoQ protein sequence from Escherichia coli.

SEQ ID NO:45—DNA coding sequence of LuxQ Receptor Histidine Kinaseinvolved in quorum sensing from Vibrio harveyi.

SEQ ID NO:46—Protein sequence of LuxQ Receptor Histidine Kinase involvedin quorum sensing from Vibrio harveyi.

SEQ ID NO:47—DNA recognition sequence for PhoB.

SEQ ID NO:48—Partial protein sequence of TrzPhoR: Trz HAMP domain+ValineA/D fusion.

SEQ ID NO:49—Partial protein sequence of PhoR histidine kinaseEscherichia coli sequence.

SEQ ID NO:50—Partial EnvZ protein sequence spanning fusion region.

SEQ ID NO:51—Partial Trg protein sequence spanning fusion region.

SEQ ID NO:52—Partial Trz protein sequence spanning fusion region.

SEQ ID NO:53—Partial PhoR protein sequence spanning fusion region.

SEQ ID NO:54—Partial protein sequence of coiled coil test constructTrg″CC″Pho.

SEQ ID NO:55—Partial protein sequence of coiled coil test constructTrzEnvZCC.

SEQ ID NO:56—Partial protein sequence of coiled coil test constructTrzEnvZcc2&3.

SEQ ID NO:57—Partial protein sequence of coiled coil test constructTrgPhoRcc3.

SEQ ID NO:58—Partial protein sequence of coiled coil test constructTrzHAMPPhoR fusion.

SEQ ID NO:59—Partial protein sequence of coiled coil test constructTrzHAMP+V PhoR.

SEQ ID NO:60—Partial protein sequence of coiled coil test constructTrzHAMP+VK.

SEQ ID NO:61—Partial protein sequence of coiled coil test constructTrgHAMP+V.

SEQ ID NO:62—Partial protein sequence of coiled coil D position mutantsTrzHAMP+M.

SEQ ID NO:63—Partial protein sequence of coiled coil D position mutantsTrzHAMP+V.

SEQ ID NO:64—Partial protein sequence of coiled coil D position mutantsTrgHAMP+V.

SEQ ID NO:65—Partial protein sequence of coiled coil D position mutantsTrgHAMP+G.

SEQ ID NO:66—Partial protein sequence of coiled coil D position mutantsTrgHAMP+A.

SEQ ID NO:67—Partial protein sequence of coiled coil D position mutantsTrgHAMP+L.

SEQ ID NO:68—Partial protein sequence of coiled coil D position mutantsTrgHAMP+I.

SEQ ID NO:69—Partial protein sequence of coiled coil D position mutantsTrgHAMP+E.

SEQ ID NO:70—Partial protein sequence of coiled coil D position mutantsTrgHAMP+T.

SEQ ID NO:71—Partial protein sequence of EnvZ (SEQ ID NO:71) spanningthe ADD residues (275-277) of EnvZ.

SEQ ID NO:72—Partial protein sequence of PhoR (SEQ ID NO:72) spanningthe EGA residues corresponding to the ADD residues (275-277) of EnvZ.

SEQ ID NO:73—DNA coding sequence of MBP 6.1-5.

SEQ ID NO:74—DNA coding sequence for TAL transcription factor engineeredto bind Gal4 binding sites.

SEQ ID NO:75—DNA coding sequence for Gal4VP64 codon optimized forArabidopsis.

SEQ ID NO:76—DNA coding sequence for Firefly luciferase codon optimizedfor Arabidopsis.

DETAILED DESCRIPTION

The present disclosure provides novel receptor fusions with thehistidine kinase PhoR for use in synthetic signaling systems with bothhigh throughput systems (e.g., bacteria) and in plants. The source ofthe receptors may be other histidine kinase receptors such as those frompathogenic bacteria, e.g., the quorum sensing receptor RpfC. Because ofthe inventors' robust abilities with computational protein design, thereceptor component can now be computationally designed. Alternatively,the receptors may be derived from chemoreceptor involved in periplasmicbinding protein (PBPs) mediated chemotaxis such as Trg or Tar. Eitherthe PBPs or the receptors have computationally re-designed bindingpockets or are able to be entirely computationally redesigned, allowingthe detection of novel, man-made substances in the environment and/ormetabolic products of pathogens and/or metabolic products of biologicalorganisms in general. The present disclosure also establishes theability to convert nonfunctioning receptor histidine kinase fusions intofunctional fusions by manipulating a specific region of the histidinekinase, the CA interacting region. One application of the presentdisclosure is for use in detecting novel environmental signals usingdetector plants enabled with the present synthetic signaling system.

Plants, bacteria, and fungi can sense aspects of their environmentthrough two-component or histidine kinase (HK) signal transductionsystems that transmit information via protein to proteinphosphoryl-transfer. Two component signal transduction systems respondto specific inputs such as the presence of ligands, osmotic conditions,oxidative conditions, or factors contributing to pathogenesis. Thesimplest two-component systems (TCS) use a plasma membrane localizedhistidine kinase and an intracellular response regulator (RR) protein.The RR functions to both receive the phosphoryl signal at an aspartateresidue and initiate a transcriptional response upon becomingphosphorylated (FIG. 1A). More complex hybrid two component systemsfound in bacteria and plants involve additional components (FIG. 1B).TCS have been used in synthetic biology and biotechnology applications,primarily as components in synthetic signaling pathways.

Typically sensing of an environmental aspect through a TCS consists ofthree distinct phases, signal transmission, signal dependent kinaseactivation and signal transduction. In signal transmission a membranelocalized receptor senses an environmental stimulus or binds to amolecular signal (ligand). The sensing event causes a conformationalchange in the receptor and the conformational change propagates acrossthe membrane (trans-membrane signaling) to the cytoplasmically localizedportion of the histidine kinase. Signal dependent kinase activationoccurs when the receptor signal acts upon a distinct region of thehistidine kinase causing a conformational change. The conformationalchange triggers a switch from a kinase “off” to a kinase “on”configuration of the HK. Activation of kinase activity results inautophosphorylation of a conserved Histidine residue. Signaltransduction occurs when a RR interacts with the HK, resulting in atransfer of the phosphoryl group from the HK to the RR. Thephosphorylated RR undergoes a conformational change which allows it toactivate transcription of genes that will allow the plant, bacteria orfungus to respond to the environmental stimulus.

The inventors previously described a synthetic two-component signalingsystem in plants based on bacterial derived histidine kinases and thebacterial RR PhoB (U.S. Pat. Nos. 8,148,605 and 9,062,320, both of whichare incorporated herein by reference in their entirety). In thissynthetic system, computationally designed receptors based on bacterialperiplasmic binding proteins bind a substance of interest e.g., theexplosive TNT. Periplasmic binding proteins (PBPs) are involved inbinding of a wide variety of substances in gram negative bacteria. Asubset of PBPs interact with the chemotactic receptors Trg, Tar, and Tapand initiate a signaling cascade (FIG. 1C) that allows the bacteria torespond to the presence of certain sugars (glucose, ribose, maltose,galactose) and amino acids (dipeptides, asp, glu, ser, ala and gly) bydirecting the cells to environments containing these nutrients. Theligand binding sites of ribose binding protein (RBP) and maltose bindingprotein (MBP) have been computationally re-designed to bind a variety ofsubstances, among them a nerve toxin agent surrogate, the metal zinc and2,4,6 trinitrotoluene (TNT) (Looger, et al., Nature 423:185-190, 2003;Feng, et al., eLife 4:e10606, 2015; Bick, et al., eLife 6; e28909, 2017;Strauch, et al., Nat. Biotechnol. 35:667-671, 2017). When a PBP bindsits ligand a conformational change is produced, leading to increasedaffinity of the PBP-ligand complex for the extracellular domain of abacterial chemotactic receptor, Trg in the case of RBP and Tar in thecase of MBP. In this synthetic signaling system a chimeric fusionbetween the chemotactic receptor Trg with the cognate HK for PhoB, PhoRallows direction of the signal through PhoB. The inventors discoveredthat PhoB translocates to the plant's nucleus in a signal dependentmanner. In this plant system PhoB has been modified to activate thetranscription of plant genes (FIG. 2 ). In one aspect of the system,this synthetic two component signal transduction pathway activates a setof genes which breaks down chlorophyll (de-greening circuit). Thisresults in the ability to produce a monitor or detector plant whichde-greens specifically in the presence of a substance of interest. Thissynthetic signal transduction system allows one to artificially controlbiological input-output in plants as well as test, monitor and perfectcomponents in bacteria.

The present disclosure exploits cells or plants' sensing mechanisms forextracellular signals, with the development of cells or plants thatrespond to a variety of biological, chemical, and environmentalpollutants for substances of interest to produce a readily detectableresponse or phenotype. In a particular embodiment the plants disclosedherein lose green color when exposed to a specific substance; thedegreening is an easily detectable biomarker and does not requiresophisticated instrumentation. The modularity of the system allows awide variety of responses, both visible and non-visible, to be producedin response to a detection event. These plants function as “sentinels”and are especially useful for widespread monitoring of substances in theenvironment whether interior or exterior.

For the plants to be useful as degreening biomarkers to detect specificchemical agents or to monitor environmental factors, an appropriateinput circuit was produced. This input circuit is useful for linkingdetection to response. The modularity of the system allows a widevariety of responses, both visible and non-visible, to be produced inresponse to a detection event. When the input circuit is linked to thedegreening circuit, a plant detector is produced. In addition, theability to control response of plants and biological organisms tospecific substances provides a useful tool for biotechnology allowing,for example, co-ordination of crop plants, facilitating harvesting andcontrolling other developmental, tissue or environmental responses.

The present disclosure provides a highly specific and sensitive methodfor cells or plants to detect a target substance of interest in theirenvironment, transmit the sensing from outside the cell or plant to thenucleus, induce a specific transcriptional response and a type of output(for example controlled degreening) that provides detection to humans.In one embodiment of the present disclosure, the regulatory circuitshave two components, referred to herein as input and output circuits. Inanother embodiment of the present disclosure, the input circuit has anability to specifically recognize (bind) the target substance ofinterest and transmit a signal to the nucleus, where a specific responseis initiated. The response can be a phenotypic and/or metabolic changeof interest or a visible response to produce a plant sentinel. Themodularity of the system allows a wide variety of responses, bothvisible and non-visible, to be produced in response to a detectionevent. In one embodiment of the present disclosure, one output circuitproduces a degreening or other detectable phenotype in the transgenicplant containing the circuits. In one embodiment of the presentdisclosure the output circuit is also modular in that a variety of genescan be placed under control of the signal-inducible promoter. In oneembodiment the input circuit is modular in that the receptor that istargeted to the extracellular space can be designed to providespecificity and selectivity for binding a given target substance ofinterest. One specific input circuit specifically exemplified hereinprovides detection of the explosive trinitrotoluene. One specific outputcircuit specifically exemplified herein serves as a simple and sensitivemarker that can easily be recognized directly (visually), or by remotesensing and/or by monitoring changes in chlorophyll fluorescence, bychanges in photosystem I and/or photosystem II, electron transport, bychanges in hyper-spectral imaging, and/or by changes in spectralproperties. The modular nature of the system provides the ability toproduce numerous types of responses or readouts including non-visiblereadouts that are detectable by webcam and high altitude platforms.

The input circuit comprises a sensor protein specifically targeted tothe extracellular space of the cell or plant with a binding sitespecific for recognizing a target agent or a target substance ofinterest, a transmembrane histidine kinase protein, a nuclear shuttlingprotein, and a synthetically designed signal responsive promoter.Variations and elaborations described herein are found in variousresearch publications, and known to those skilled in the art. One typeof output circuit described herein activates the expression of one ormore genes, which results in a degreening phenotype in transgenic plantscontaining the circuits.

The present disclosure provides a sensor protein or receptor at the cellsurface, such that the sensor protein or receptor has a binding sitespecific for the target substance of interest. The transmembraneprotein, a second component of the input circuit has three parts: aninteracting domain, a transmembrane domain and a histidine kinasedomain. Binding of the target substance of interest causes aconformational change in the sensor protein or receptor, so that it thenbinds to an interacting domain of the transmembrane protein on theexterior surface. The interaction of the sensor protein:target substanceof interest complex results in activation of the histidine kinase,typically by an autophosphorylation mechanism. The interaction of thesensor protein or receptor with the interacting domain produces aconformational change in the transmembrane protein and/or transmembranehistidine kinase. The autophosphorylated histidine kinase domain of thetransmembrane protein transfers a high energy phosphate group to acytoplasmically located protein. A variety of proteins will function asshuttling proteins, including, but not limited to, a syntheticallyadapted shuttling protein such as PhoB:VP64, other shuttling proteinssuch as histidine phosphotransferases, Arabidopsis histidinephosphotransferase, and other natural proteins such as responseregulators from plants, bacteria, fungi, and cyanobacteria systems,including adapted or synthetic proteins, or computationally designedproteins, that function in histidine kinase mediated signaling systems.

The shuttling protein typically has several functions includingreception of the signal from the transmembrane protein, relay of thissignal to the nucleus, or specific responding component, and/oractivation of transcription. The protein may directly, or indirectly,bring about a cellular response. The typical cellular response isactivation of transcription; however, other responses are possibleincluding changes in membrane potential, cell expansion (in the case ofengineering a response that would allow expansion of the xylem), orchanges in the accumulation of a plant-derived product. At least someproteins are phosphorylated (directly) by the histidine kinase domain ofthe transmembrane protein. The phosphorylation of the proteins orprotein components can cause an increase in binding affinity for aspecific sequence of DNA as is the case for OmpR, or in the case ofPhoB, allow a conformational change that removes repression, allowingthe DNA binding domain to function. One type of response of this is areadout circuit that includes expression of the specifically regulatedgene located in the nucleus of the plant and the production of adetectable phenotype, appearance or function of lack thereof or thereadout can include activation of a gene controlling a trait ofinterest, for example, flowering or ripening.

The sensor protein or receptor can be derived from a bacterial (e.g.,Escherichia coli) periplasmic binding protein (PBP), such as a maltose,ribose or galactose PBP, and the binding site for the target substanceof interest can be a naturally occurring binding site or one that is theresult of computational design. At the N-terminus there is also a signalpeptide sequence for targeting the sensor protein to the exterior of thecell, plant or plant cell, such as, but not limited to, the signalpeptide of the pollen PEX protein (Baumberger, et al., Plant Physiol.131:1313-1326, 2003). Substances of interest can include, withoutlimitation, plant hormones, explosives, chemical agents, products ofindustrial manufacturing, metabolites of biological organism(s),environmental pollutants including all currently listed environmentalpollutants on the Environmental Protection Agency (EPA) superfund site,halogenated hydrocarbons, or degradation products, metal ions such aszinc, a heavy metal, a sugar, neurotransmitter, herbicides, pathogenicproducts, or an amino acid. The ability to computationally design thePBP, partially or entirely, expands this list of detectable substancesto most molecules.

When the target substance of interest is bound to the sensor protein orreceptor, there is an interaction with the protein that transmits asignal from the exterior of the plant to the interior of the plant byautophosphorylation and activation of the histidine kinase. Upon bindingof the target substance of interest, there is an interaction between orwithin the sensor protein or receptor and the transmembrane protein(which contains the histidine kinase domain). This interaction causesautophosphorylation of a histidine residue located on the interiorportion of the transmembrane protein. The phosphoryl group is thentransferred (a mechanism called phosphor-relay or phosphotransfer) to ashuttling protein or transcription activator protein domain, allowing itto translocate to the nucleus or otherwise initiate a response. Thephosphorylated protein, protein domain or secondary protein then binds aDNA recognition sequence present in a promoter of a gene (or genes) inthe nucleus, which can be a genetically engineered gene, with the resultthat transcriptional expression of that gene occurs.

The transmembrane protein can be genetically engineered as atranslational fusion consisting of plant and/or bacterial proteins,derived from one or more bacterial or plant proteins, derived from oneor more proteins containing histidine kinase-like features, orsynthetically synthesized features, or computationally designedfeatures, provided that it functions in plants in conjunction with aprotein or protein domain to transmit the signal to a response unit. Theintracellular receptive protein or protein domain can be a plantprotein, a bacterial protein or a synthetically designed protein, withthe proviso that it receives the signal from the transmembrane protein.The receptive protein can either transmit the signal to another proteinthat initiates a response, or translocate to the nucleus in response tothe signal. In one example, the signal receptive protein itself moves tothe nucleus, binds DNA and activates gene expression. Specific examplesinclude a plant histidine phosphotransferase or a bacterial protein suchas the E. coli proteins OmpR or PhoB. Where the signal receptive proteinis also a transcriptional activation protein, PhoB, the DNA recognitionsequence is CTGTCATAYAYCTGTCACAYYN (SEQ ID NO:47), and it can occur from2 to 12 times, for example 4 or 8 times in the region upstream of thetranscription start site, and includes a plant transcriptional startsite such as defined by a minimal transcriptional promoter.

The sequence that is expressed in response to detection of, or thepresence of, a target substance of interest in the plant environment canbe a protein coding sequence or it can be a functional nucleic acidsequence (such as a RNA interfering molecule, diRNA or an antisense RNAto inhibit synthesis of a related coding sequence) or it can be acombination of these. The associated expressed sequence can be a plantgene that is, in nature, expressed constitutively or in a tissue orcondition specific fashion, but in the present disclosure, it isexpressed when the target substance of interest or substance that bindsto the sensing protein or sensing proteins is present or after thetarget substance of interest is present. The expressed sequence can bevirtually any sequence of interest: a detectable marker such as green oryellow fluorescent protein or another fluorescent protein,β-glucuronidase or β-glucosidase, among others, a positive regulator offlowering or a sterility protein preferably selectively expressed in theappropriate tissue, a bioremediation coding sequence such as mercuryreductase, a phytochelatin or metal sequestering protein, an enzyme fordetoxifying a contaminant or harmful material, and the production of aspecific nutritive or pharmaceutical substance, among others. Theexpressed sequence can also be a functional nucleic acid (antisense ordiRNA to inhibit expression of a related nucleic acid sequence). Therecan be more than one target substance-regulated gene within a singlecell or plant and more than one readout or response in a single cell orplant.

In an embodiment of the present disclosure, the sensing circuitry can beused to control features of interest, such as the timing of flowering ofa plant or ripening of a fruit such that harvesting is moresynchronized, coordination of crops such as cotton, soybean and corn andhence an ability to predict harvest time, and thus, make harvesting moreefficient and economical or so that plants are in flower for aparticular occasion. Such a gene or response unit is operably linked toa promoter containing the recognition sequence of the specific sensingsystem or systems. In another embodiment, the target substance ofinterest-dependent transcription regulatory system can be used to renderplants exposed to the target substance sterile, when a sterilityinducing protein is expressed under the regulatory control of thecontrol system of the present disclosure.

Within the scope of the present disclosure are one or more DNAconstructs containing a plant operable sensor protein as describedabove, a plant transmembrane protein, a plant operable signal receptionand/or transcription activation protein that is activated by thehistidine kinase portion of the sensing circuit (via an intermediaryendogenous protein, or directly by the membrane bound kinase), and aplant operable sequence operably linked to transcription regulatorysequences, which include the recognition sequence of the particulartranscription activating protein of the disclosure. Similarly, thepresent disclosure provides transgenic plant cells, transgenic plantparts, transgenic plant tissue and transgenic plants containing one ormore constructs of the present disclosure.

The present disclosure provides transgenic (sentinel) plants useful forenvironmental monitoring and for detecting particular biological andchemical agents, environmental pollutants, and/or a specific substancesuch as herbicides or trigger compounds. Trigger compounds aresubstances that bind to the natural or computationally designed sensorproteins and thereby increase the sensor proteins affinity for anextracellular protein domain (for example Trg). In a specificembodiment, the plants disclosed herein lose green color within hours ofexposure to particular target biological/chemical agents orenvironmental pollutants. The loss of green color (or a change in thefluorescence of chlorophyll or a change in photosynthetic electrontransport, or other types of responses) in plants are easily detectable,either by direct observation, with simple hand-held machines, orremotely by aircraft, satellite, or other varieties of sensors,including webcams and high altitude platforms. The sentinel plants ofthe present disclosure comprise genetically engineered DNA constructsthat direct the expression of both the input and output circuits, asdescribed herein, with the result that the plants lose color, orotherwise respond, when they “sense” the presence of the targetsubstance of interest. An important advantage of the degreening systemin these sentinel plants is that they are capable of regreening. Theyeither regreen naturally or at an enhanced rate with treatment ofhormones, i.e., the sentinels can be reset for renewed surveillance forthe target substance to which they respond. In one aspect of the presentdisclosure a transgenic plant wherein degreening has occurred due to thepresence of a target substance of interest is able to regreen afterremoval of the external target substance of interest. Other responses orreadouts allow rapid response time or ability to detect signals remotelywith webcams or high altitude platforms.

The transgenic plants (sentinel plants) of the present disclosure can beindoor plants, for example, any of a number of species that are commonlyused as decorative accent plants, such as peace lily (Spathiphyllum),philodendron, pothos (Epipremnum), spider plant (Chlorophytum),Tradescantia and Dracaena, and the like. In addition, the sentinelplants can be crop plants such as corn, wheat, soy, cotton, soybeans andothers, or they can be grasses or trees, either deciduous (poplars,aspens, maple, oak, cottonwood, and the like) or evergreen (pines,spruce, junipers and the like) or they can be annuals or perennials usedin various types of plantings, or they can be a variety of nativespecies, or they can be aquatic plants including, but not limited to,algae. Nearly all plants and/or plant cells can be readily transformedand transformed seed directly formed or plants produced from thetransformed cells, as is well known to the art. The sentinel plants ofthe present disclosure can provide a warning of current presence of atarget substance of interest or they can provide notice to responders toa scene to allow for appropriate protective measures and/or to preventexposure to a dangerous condition. In addition, the sentinel plantsprovide the ability to remotely monitor for the presence of substances.Moreover, the sentinel plants allow for continuous environmentalmonitoring over extremely large scales (e.g., hundreds or thousands ofsquare kilometers) that is not currently possible with any otherpublicly known method.

The sentinel plants of the present disclosure contain a geneticallyengineered signaling pathway consisting of two functional parts referredto herein as “input” and “output” wherein one embodiment of the outputis the “degreening” circuit”. The input gene circuit is a natural orgenetically engineered system, or computationally designed system, thatrecognizes a biological or chemical agent, explosive, or anenvironmental pollutant or target substance of interest specifically andselectively, then activates an output gene circuit that results in thedesired response. In the case of a plant sentinel, one example of anoutput gene circuit is the degreening circuit, so that the degreeningphenotype i.e., white plants, are produced in response to an agent orpollutant. The degreening can be visually detected as a loss of greencolor or it can be detected as a change in chlorophyll fluorescence orin photosynthetic electron transport or it can be detected with avariety of spectroscopic methods such as hyper-spectral imaging andother methods. Other responses or readouts allow rapid response time orability to detect signals remotely with webcams or high altitudeplatforms.

The output and input circuits of the present disclosure are generated byexpressing DNA constructs specifically designed to provide a functionalsystem. Examples of methodologies well-known to people in the fieldinclude CRISPR-Cas9 methods, where the endogenous genes can be changedto produce plants with these properties. The input circuit is a systemcomprising a receptor or a binding protein designed to recognize (e.g.,by binding) a signal (e.g., analyte or ligand), and this binding eventultimately activates a response, one of which is transcription of a geneof the output (degreening) circuit to produce a plant sentinel. Thus,the specificity and selectivity of a given response is determined by theinput circuit. An example of the input circuit is a receptor or bindingprotein (sensor protein) that specifically binds a particular explosive,chemical agent or a pollutant, or pathogen or pathogen product, thetarget substance of interest, which, upon binding of such explosive,agent or pollutant, or pathogen or pathogen product, can transmit asignal via the transmembrane protein to activate transcription of agene(s) in the output circuit. As specifically exemplified the sensorprotein:target substance complex interacts with the exterior domain ofthe transmembrane protein, with the result that the histidine kinasebecomes active.

In one method, the response system (output, as exemplified bydegreening) circuit is generated by transforming a plant with DNAconstructs (i.e., expression vectors) comprising one or more nucleicacids encoding, or complementary to, a nucleic acid encoding key enzymesor functional fragments thereof in chlorophyll biosynthesis and/ordegradation pathway under the control of a promoter that responds to asignal from the input circuit. The term “functional fragment” as usedherein, is intended to indicate that the product (i.e., enzyme) can be atruncated protein as long as it retains its enzymatic activity to causedegreening (chlorophyll degradation). One skilled in the art would knowthat a truncated protein may be able to maintain enzyme activity.Examples of chlorophyll degradation enzymes include, but are not limitedto, RCCR, PaO and chlorophyllase. The output/degreening circuit can alsocomprises a target-substance-regulated inhibition of chlorophyllbiosynthesis. As specifically exemplified, this is achieved byexpression of either antisense, or preferably, interfering RNA molecule(such as diRNA, siRNA) sequences specific to a coding sequence for anenzyme in the chlorophyll biosynthetic pathway. These interfering RNAmolecules are examples of functional nucleic acids, and in the contextof inhibition of gene expression, a functional fragment of a codingsequence or gene is one that specifically interacts with a transcript ofthe coding sequence or gene so as to reduce expression of the product ofthat gene or coding sequence. Examples of the enzymes involved inchlorophyll biosynthesis include, but are not limited to,protochlorophyllide oxidoreductase (POR), GUN4, other GUN genes (genomeuncoupling), Mg chelatase and chlorophyll synthetase. It is understoodthat other targets in the chlorophyll synthesis or degradation pathwaycan be substituted for those specifically set forth. It is furtherunderstood that the input system allows a wide variety of outputs,responses or readouts, as long as the output, response or readout isoperationally linked to the input. These include rapid response time orability to detect signals remotely with webcams or high altitudeplatforms.

The DNA construct for transforming the readout or degreening genecircuit into a plant or plant cell typically contains a nucleic acidencoding at least one chlorophyll degradation enzyme (or a fragmentthereof that functions to effect chlorophyll degradation) and/ordesirably also a nucleic acid whose expression product inhibitschlorophyll synthesis operably linked to a promoter with transcriptionregulatory sequences that bind a transcription activator protein thatreceives the signal from the input gene circuit. Typically it can be atranscriptional activator protein that solely receives the signal fromthe transmembrane histidine kinase and shuttles to the nucleus or anuclear localized transcriptional activator protein that receives thesignal from the transmission protein that relays the signal from thetransmembrane histidine kinase and shuttles to the nucleus. The exteriorcomponent of the transmembrane histidine kinase has bound the sensorprotein substance complex therefore relaying an input signal generatedby an explosive, a chemical or biological agent, a pollutant, a pathogenor pathogen product, or a specific substance. In response to the inputsignal, this dual modulation, i.e., inhibition of synthesis andstimulation of degradation of chlorophyll ensures loss of green color inplants when exposed to a variety of chemical agents or environmentalpollutants. As described herein, chlorophyll synthesis can be inhibitedby producing interfering RNA or antisense RNA derived from at least oneof the genes encoding chlorophyll synthetic enzymes.

Accordingly, a transgenic plant containing the input and output circuitsdisclosed herein loses its green color when exposed to a substance inthe environment that activates the input circuit by binding to aspecific receptor site (i.e., sensor protein) outside the plant. Thesubstance can be, for example, a chemical agent, mercury, lead, arsenic,uranium, cadmium, selenium, polycyclic aromatic hydrocarbon, a benzene,a toluene, a xylene, or a halogenated (chloro, fluoro, and chlorofluoro)hydrocarbon, a by-product of industrial manufacturing, a metabolite ofbiological organisms, explosives, any substance listed on the EPAsuperfund website, specific compounds involved in manufacture ofcompounds of interest, a pathogen or pathogen product, or a triggersubstance to bring about a desired change in the plant or crop. It isalso possible to wire the genetic circuitry to enable detection ofmultiple substances. In addition, the target substance that binds to aspecifically engineered sensor protein and input circuit via theextracellular receptor could be an explosive such as trinitrotoluene,other types of explosives, or a degradation product of one of theforegoing compounds specifically bound by the sensor protein, includingcomputationally designed sensor proteins.

The sensing and response system of this disclosure is modular in that itcan be coupled with a variety of input circuits (sensor proteins) toprovide specificity and selectivity for a particular chemical agentand/or other environmental factor of interest that is recognized by anavailable sensor protein that effectively interacts with the exteriordomain of the transmembrane protein when the target substance is bound.Likewise, the input circuits can be combined with Logic Gates (e.g.,AND, OR, NOT gates organized, for example, as detect substance “a” ANDdetect substance “b”; detect substance “a” OR substance “b”; detectsubstance “a” OR substance “b” but NOT substance “c,” etc.) to furtherincrease the present technology's uses. Similarly, the readout gene thatis expressed via the histidine kinase system or systems of thisdisclosure can be selected for a desired result, with the proviso thatit is operably linked to a promoter and associated control sequencesthat interact positively with a transcription regulatory proteinactivated directly or indirectly by the histidine kinase and/or PhoB orOmpR, described herein. Specifically, receptors that are engineered tobind site specifically to the target substance of interest (includingbut not limited to heavy metals, chemical agents, explosives and certaindegradation products thereof, environmental pollutants such as MTBE,herbicides such as glyphosate and the like. The sensing circuit furtherincludes the transmembrane protein with an external binding domain thatinteracts with the sensing protein-target substance complex and anintracellular portion which directs the phosphorylation of atranscriptional activator protein, as specifically exemplified by PhoBand/or modified and/or an adapted version of the PhoB protein. PhoB canalso be phosphorylated by an endogenous plant histidinephosphotransferase. The phosphorylated PhoB (activated form) then bindsto the PhoB cognate binding sequences which are part of the syntheticpromoter operably liked to a chlorophyll degradation enzyme codingsequence (such as chlorophyllase). The transcriptional activator proteincan also be a hybrid protein including, but not limited to, PhoB:VP64translational fusion protein and it is expressed in a transgenic plantexpressing its coding sequence operably linked to a plant expressiblepromoter, which can be constitutive or which can include sequences fortissue-specific or condition-specific expression. The activator proteincan be any eukaryotic transcriptional activator including, but notlimited to, VP16, VP64 and GAL4. The signal-dependent nucleartranslocating PhoB protein could also be fused to synthetic repressors,including LexA-EAR (LEAR) or Gal4-EAR (GEAR) (U.S. Patent ApplicationPublication Number 2018/0105825), or OfpX (Wang, et al., PLoS One6:e23896, 2011).

Histidine Kinase Signal Transduction System

Two component histidine kinase signal transduction systems are conservedbetween plants and bacteria, and this conservation was the basis offorming a functional input (sensing) circuit. In bacteria, sensitivechemotactic sensors exist to direct motile bacteria to nutrients, e.g.,ribose. When a periplasmic binding protein such as the ribose bindingprotein binds its ligand, it develops a high affinity for theextracellular domain of bacterial chemotactic receptors such as Trg.Upon binding of the ligand/binding protein complex, a cytoplasmichistidine kinase is activated. Normally in the bacterium, this resultsin chemotaxis toward the food source. Hybrid histidine kinases have beenexpressed in bacteria where the cell surface PBP binding domain of Trghas been combined with the interior histidine kinase domain fromproteins such as EnvZ. This hybrid protein activates transcription viaphosphorylated transcription activator proteins. In the hybrid histidinekinases, the target substance is bound by the sensor protein, and thesubstance:protein complex binds to the interacting domain of the hybridhistidine kinase at the exterior side of the cell membrane, and thatinitiates activation of the histidine kinase (HK). The HK starts aphospho-relay (phosphorylation relay) through a bacteria responseregulator (e.g., OmpR or PhoB) to activate transcription of bacterialgenes. The phospho-relay always goes His→Asp→His, etc. In addition, atleast some transcription activator proteins are phosphorylated(activated) by that same kinase domain.

Chemotactic binding proteins (periplasmic binding proteins) have beenredesigned using computer-run computational design methods so thatinstead of binding substances such as ribose or galactose or maltose,the engineered proteins specifically bind a target substance of interestsuch as TNT, chemical agents, heavy metals, or other environmentalpollutants or harmful substances.

Plants also use a two-component or histidine kinase signaling systemthat responds to cytokinin (a plant hormone). Plant signal transductionis more complex. The histidine kinases are “hybrid types”. The plant HKsin Arabidopsis are known as AHKs. Upon sensing cytokinin, plant HKsphosphorylate an internal histidine kinase and initiate a phospho-relayinternally to an aspartate residue located in the receiver domain of thesame protein. The receiver domain transfers the phosphate group to anindependent protein. The independent protein moves into the plant cellnucleus upon phosphorylation and then transfers the phosphate group to anuclear localized protein, ARR Type B, transcription factors that theninitiate transcription of ARR Type A genes. Examples of ARR type A genesuseful in the present disclosure include, but are not limited to, ARR5and ARR7, or any Type A ARR gene. Other functionally equivalentsequences may also be used in the systems described herein.

Computer design enables the design of sensor proteins to bind with greatspecificity and sensitivity, a variety of compounds or substances. Inbacteria, the engineered receptors were targeted to the periplasmicspace to sense various substances of interest. In plant cells, it isnecessary to add (desirably at the N-terminus) a secretory sequencefunctional in plant cells so that the sensor protein is at the exteriorof the cell and can bind the particular target substance of interest andit is necessary to delete the bacterial periplasmic targeting leader.The starting point is the engineered periplasmic binding protein, andthe ending point is a detectable change resulting from a transcriptionalresponse in the nucleus; computer-designed sensor proteins and molecularbiological techniques allows for the combination. Hybrids at both thestarting point and ending point allowed functional signaling.

To obtain information from outside the plant cell and transmit a signalto the nucleus of the plant cell, specifically engineered target sensingreceptors were positioned outside of plant cells. Receptors that havebeen computationally designed include, but are not limited to, theperiplasmic binding proteins RBP, MBP (maltose binding protein) and GBP(galactose binding protein). Importantly, at least in part because thesystem is modular, PCR or DNA synthesis can be used to change thereceptor/sensor protein portion from a receptor/sensor protein specificfor TNT to a target substance of interest (explosives, chemical agents,zinc, heavy metal, environmental pollutant).

Plant Extracellular Space

Plants are not known to have a functional periplasmic space. However,evidence indicates that there is a functional space between the plantplasma membrane and the outside. Small proteins can freely move and/ordiffuse in the plant cell wall, better understood as a complex matrix,and even move and/or diffuse in the plant cuticle, the waxy coating thatis found outside some plant organs. In bacteria, the periplasmic bindingprotein contains a leader peptide portion that targets the protein tothe periplasm. In plants, proteins are targeted to the extra-cellularspace by way of the endoplasmic reticulum. Because of the differenttargeting mechanisms, a plant extracellular targeting sequence is neededand the bacterial periplasmic targeting leader must be removed.

Genetically Engineered Plants Capable of Losing Green Color

The present disclosure also provides genetically engineered plantscapable of losing green color in response to a signal (analyte orligand) by simultaneously controlling expression of genes involved inchlorophyll biosynthesis and/or degradation. These plants are capable ofreceiving input from cytoplasmic and extracellular analytes and linkingthese components to the degreening circuit resulting in the loss ofgreen color. Thus, the plants of this disclosure serve as a simple andeasily detectable biomarker for adverse environmental input.

The degreening circuit is assembled in a “plug and play” manner. Hence,the sensor protein for TNT, which initiates the input, can be replacedby a different computationally designed sensor protein allowing thedegreening circuit to respond to a specific target substance or targetsubstances of interest. The model plant species Arabidopsis, whichallows rapid optimization of the degreening circuit and its response,can be used in the presently disclosed compositions and methods.However, the circuits described herein are readily introduced into otherplant species such as those typical of shopping malls, office buildings,landscapes, forested areas, cropland or aquatic systems.

The plants of this disclosure that lose their green color in response toa target substance can serve as untiring sentinels reporting on adverseinput from the environment (e.g., chemical weapons or pollutants). Plantsentinels would be unthreatening to the general public and can bedeployed in shopping malls and office buildings and at special eventswhere most people can recognize a loss of green color and securitypersonnel could easily detect the changes within a short period withinexpensive hand-held machines. In addition, loss of green color orother disruption of chlorophyll, such as chlorophyll fluorescence, orphotosystem electron transport or hyper-spectral imaging can be rapidlyquantified by authorities with either portable hand-held equipment orsimple laboratory equipment (spectrophotometers). In vast geographicareas, detector systems could be introduced into plants typical forlandscapes and aquatic systems, allowing satellites to identify adverseenvironments.

The degreening circuit of the present disclosure induces genes that areinvolved in chlorophyll breakdown and synthetic genes for inhibitingchlorophyll synthesis. Simultaneous expression of the genes thatinitiate chlorophyll breakdown and inhibit new chlorophyll biosynthesiswould yield the most efficient degreening phenotype. For this reason,the degreening circuit can be created using three genes, two in thechlorophyll degradation pathway and one inhibitory gene in thechlorophyll biosynthesis pathway. A person of ordinary skill in the artunderstands that other combinations of the genes that are known to beinvolved in chlorophyll synthesis and degradation can be used to obtainthe degreening phenotype demonstrated herein. In addition, a person ofordinary skill in the art understands that the reactive oxygen species(ROS) generated in the chloroplast could be used to initiate andgenerate the degreening within plastids.

The degreening circuit of the present disclosure can respond in twodifferent ways; it can respond to target substances within the cytoplasmas well as those that are extracellular. To test the ability of thedegreening circuit to function with cytoplasmic input in plants, asynthetic cytoplasmic receptor is linked to the circuit. In response tobinding an analyte, the cytoplasmic receptor is transported to thenucleus where it activates synthetic transcriptional promoter(s) fusedto genes whose products degrade chlorophyll while preventing newchlorophyll biosynthesis. To test the ability of the degreening circuitto function with input from outside the plant, an input circuitcontaining a chimeric receptor or binding protein can be linked to thedegreening circuit. In response to binding an analyte, the extracellularreceptor initiates a signal transduction pathway and activates a signalreceptive synthetic transcriptional promoter fused to genes whoseproducts degrade chlorophyll while preventing new chlorophyllbiosynthesis.

Normal time periods for notable loss of green color in plants varieswidely from days to weeks depending on whether the loss is triggeredfrom environmental changes, development (e.g., flower petals) or stress(e.g., pathogens). To develop a system that can lose green color rapidlyin response to a signal, both the chlorophyll biosynthesis andchlorophyll breakdown pathways were modified to construct a “degreeningcircuit”. In addition to genes involved in chlorophyll metabolism, aredundant marker, green fluorescent protein (GFP) can be included in thedegreening circuit as a control. The GFP marker is similarly(optionally) linked to the input part of the circuit and serves toeliminate false positives that might arise.

To ensure that the degreening phenotype appears rapidly, two genes (forexample, chlorophyllase and RCCR) were used in the degreening circuitexemplified herein. Although it was not measured, the turnover inchlorophyll is strongly believed to have stimulated feedback inductionof new chlorophyll biosynthesis. To prevent this from occurring in thedegreening circuit, expression of the protochlorophyllide oxidoreductase(POR) gene, the rate-limiting enzyme in chlorophyll biosynthesis, wasinhibited.

One approach to prevent expression of (silence) a specific gene involvesthe production of an interfering RNA molecule that contains a sequenceidentical to the gene of interest. Typically, the plants are geneticallyengineered to express inverted repeats (500-700 bp) to the gene ofinterest. The resulting double-stranded RNA is homologous to anendogenous transcript. Transgenic plants containing diRNA show highturnover rates of the homologous transcript and complete silencing ofthe endogenous gene expression. An interfering RNA molecule has beenshown to be more efficient than antisense RNA in blocking the expressionof a desired gene with silencing frequency between 90-100%. Thus theinitial degreening circuit was generated using double stranded RNAs tosilence the POR gene in a transgenic plant and hence prevent the de novosynthesis of chlorophyll after input from an analyte. A series ofconvenient Arabidopsis vectors for making dsRNA constructs are publiclyavailable. These vectors contain a cassette for cloning a desired geneor gene portion in the sense and antisense orientations. The cassettehas two pairs of unique restriction enzyme recognition sites flanking a335 base pair GUS (0-glucuronidase) fragment that separates sense andantisense regions of the inverted repeat and facilitates formation ofthe dsRNA. The vectors are a series of plasmids that replicate in bothE. coli and Agrobacterium tumefaciens allowing easy cloning and planttransformation, respectively. Vectors are available carrying the Bar orNptII genes, the plants containing the introduced genes can be selectedwith the herbicide BASTA (glufosinate ammonium) or the antibiotickanamycin, respectively. A chloamphenicol or spectinomycin gene providesbacterial selection. For example, the conserved region ofprotochlorophyllide oxidoreductase (POR) gene can be cloned in the senseand antisense direction to produce the diRNA molecule specific for thePOR genes. The vectors are designed to direct expression of the diRNAmolecule with a strong constitutive promoter (CaMV 35S). To place thediRNA vector in the degreening circuit, this promoter, which is flankedwith unique restriction sites, is replaced with promoters that placeexpression under control of perception of cytoplasmic or extracellularanalytes for example, using the Pho promoter described.

Assembly and Testing of Degreening Gene Circuits

In many biological responses, sensing of a specific substance leads to atranscriptional response. The synthetic sensing system for plantsentinels links input to transcriptional output, hence, a test readoutsystem was created that is triggered by a transcriptional response(signal-regulated induction of gene expression). Numeroustranscriptional induction systems are available that provide a model inwhich to test the chlorophyll reporter system. A synthetically designed,steroid inducible system was modified to function in plants. In thepresence of a synthetic steroid (4-hydroxytamoxifen, 4-OHT), a chimerictranscriptional regulator relocates to the nucleus and inducesexpression of a promoter made up of specific response elements and the−46 region of the CaMV35S promoter, designated 10XN1P. The 4-OHTinduction system is essentially analogous to other transcriptionalinducible systems.

In order to use plants to monitor large areas for pollution or terroristagents, a reporter or readout system is needed. Prior gene reportersystems were developed for laboratory use and do not providecharacteristics needed for a plant sentinel. A synthetic degreeningcircuit was developed that allows the green pigment chlorophyll to beused as a biosensor readout system. Induction of the degreening circuitallows remote detection, displays a rapid response, provides a resetcapacity, and results in a phenotype readily recognized by the generalpublic. Because the degreening circuit produces a white phenotype, it iseasy to distinguish it from plants stressed from biotic or abioticconditions, which produce yellow (or other color) phenotypes viasenescence-related pathways. The inability to reset biosensors has beenthe major limitation to their use. The degreening circuit provides asimple capacity to be reset. Plants regreened after removal of theinducer, and this regreening was enhanced by a brief cytokinintreatment. Because the transcriptional inducer used (4-OHT) isrelatively stable, the degreening circuit may not fully switch to an“off” position immediately following removal of the inducer, and theregreening process may not start until the inducer within the plantdegrades. Hence, it should be possible to substantially reduce the timeneeded for regreening, currently about 3 days.

The degreening circuit, combining “stop-synthesis” with an “initiatebreakdown” function, caused loss of chlorophyll with unprecedentedspeed. When each function was introduced separately, plants did notvisibly degreen in the 48 hour timeframe except in the cotyledons.Expression of the “initiate degradation” circuits (CHLASE and PAO, orCHLASE and RCCR) failed to produce rapid degreening, suggesting thatplants can enhance chlorophyll biosynthesis when needed. Likewise, the“stop synthesis” circuits (diRNA specific to POR or GUN4) failed toproduce rapid degreening, supporting the concept of a large amount ofmetabolically stable chlorophyll within the plant. The rationalcombination of these two functions in one T-DNA construct produced asynthetic “degreening circuit”. The designed gene circuit is successfulwith respect to signal responsiveness, as indicated by three types ofdata: response of excised leaves to dark-induced senescence, distinctiveultrastructural changes, and microarray data showing a difference ingenes regulated by the degreening circuit and normal chlorophyll loss insenescence.

Light was shown to be important for the rapid degreening process tooccur, as induced plants incubated in the dark failed to turn white,even after 72 hours of induction. When induced plants were transferredto light, degreening proceeded at an enhanced rate. These resultssuggest that the degreening circuit is poised to respond in darkness,but not able to initiate rapid degreening without light. Chlorophyllbiosynthesis and breakdown intermediates are potentially phototoxic.Because the degreening circuit interferes with the normal balance ofchlorophyll and likely its metabolic intermediates, it is possible that,upon light exposure, these molecules cause photo-oxidation of pigments.A similar light requirement for degreening was observed for detachedleaves. Under standard light conditions degreening induction causeddetached leaves to fully degreen within 48 hours. However, darknessfailed to induce full degreening in detached leaves, even after 72 hoursof induction. Because darkness has been shown to induce senescence inArabidopsis detached leaves, these results suggest that chlorophyll lossfrom the degreening circuit is distinct from senescence.

The degreening circuit provides an effective means to controlchlorophyll levels in plants. The trigger for the degreening circuit isa specific input, resulting from sensing of the binding of a targetsubstance of interest outside the plant, with signal transduction viahistidine kinase within the cell and nuclear transcription activation.The steroid-inducible 10XN1P promoter used with the degreening circuitas a model can be replaced with other promoter elements, such as thoseresponsive to signal transduction or the synthetic PlantPho promoter, asreadily understood in the art. By combining the controlled chlorophyllloss as a reporting element with a sensing system such ascomputationally designed receptors or sensor proteins that provide inputvia transmembrane histidine kinases, plants are produced to serve asinexpensive monitors for terrorist agents, environmental pollutants orother target substances of interest. Degreening indicating presence ofthe target substance can be observed visually at close range or detectedfrom a distance by remote sensing, as known to the art.

All DNA constructs, transgenic plant cells, tissue and plants, andmethods for detecting a target substance of interest or for obtaininggene expression in response to the presence of the target substance ofinterest are within the scope of the present disclosure. It is furtherunderstood that other evolutionarily conserved signal transductioncomponents and systems, and transcription regulatory components can besubstituted for those recited herein, provided that there are functionalinput and/or output circuits responsive to the presence of a targetsubstance.

Definitions

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe present disclosure.

The term “transgenic plant” is used herein to indicate a plant, orphotosynthetic organism including algae, that has been geneticallymodified to contain exogenous or heterologous DNA to obtain a desiredphenotype. Examples of the exogenous DNA molecules that have beentransformed into the plants of the present disclosure include thoseencoding segments of DNA encoding the sensor protein, the transmembraneprotein, a shuttling and/or response protein, and a receptive promoter,collectively known as the response circuits and/or those encodingsegments of chlorophyll biosynthetic and/or complete degradation enzymesand a promoter that is responsive to a signal.

The term, “plant,” as used in the present disclosure, is intended tocover any plant, vascular or nonvascular, aquatic or terrestrial; algae,and organisms formally and informally recognized as algae now moreproperly known as cyanobacteria are included within this definition.

The term “non-plant organism” includes, but is not limited to, Archea,bacteria, fungi including yeast and cyanobacteria and the like and otherorganisms containing two-component signaling systems.

The term “degreening,” also referred to as a “loss of green color,” isintended to indicate a loss of chlorophyll and photosynthetic pigmentsin the transgenic plants that is distinguishable from normal plants(non-transgenic plants). The degreening can be detected visibly, or witha variety of instruments that measure properties including, but notlimited to, chlorophyll fluorescence, hyper-spectral imaging, infra-redand near-infra-red imaging, multi-spectral imaging, photosyntheticproperties and properties related to reactive oxygen species and theirdamage. The measurement instruments can be hand-held, or instrumentsthat function at a distance, the distance being from aircraft orsatellites.

The term “external signal,” or “environmental signal,” or “targetsubstance of interest,” is intended to mean a signal typically in theform of an analyte or ligand that triggers the signaling pathway in thetransgenic plants of the present disclosure and results in thedegreening phenotype and/or a change such as induction of geneexpression of interest. In this sense, the signal can be any biologicalor chemical agent including environmental pollutants. The substance canbe, for example, sugars, herbicides, a poison, a pollutant, a toxin,heavy metals such as mercury, lead, arsenic, uranium, cadmium, selenium,polycyclic aromatic hydrocarbon, a benzene, a toluene, a xylene, or ahalogenated (chloro, fluoro, and chlorofluoro) hydrocarbon, a steroid orother hormone. In addition, the target substance that binds to aspecifically engineered input circuit via the extracellular receptorcould be an explosive such as TNT (trinitrotoluene) or other explosive,or a degradation product of one of the foregoing compounds recognized bythe input circuit via specific receptor site binding by the sensorprotein. Any target substance for which a sensor protein can becomputationally designed (Looger, et al., Nature 423:185-190, 2003;Dwyer, et al., Curr. Opin. Struct. Biol. 14:495-504, 2004) can serve asan external signal in the context of the present disclosure.

The term “detectable marker” is a change brought about in a plant thatis perceivable or capable of being sensed by humans, other organismssuch as, but not limited to, dogs, and/or machines. The change can bevisible or invisible to humans. The sensing can involve non-destructive(for example, multi-spectral imaging) or destructive methods (forexample, analysis of protein, DNA, RNA or metabolic product).

The term “response regulator domain” is a protein or portion of aprotein that contains conserved amino acids collectively functioning toperceive a phosphor-relay from an activated histidine kinase. Theconserved domain may contain a phosphor-accepting Asp or His residue, orit may contain other residues that can be made capable of accepting theactivated phosphate.

The term “response gene” is a gene whose expression is linked to inputfrom the sensor protein or proteins.

The term “sensor protein” is used interchangeably with “receptor.”

The term “transmembrane protein” is used interchangeably with “histidinekinase”.

The terms “expression construct” or “DNA construct” are usedinterchangeably herein and indicate a DNA construct comprisingparticular sequences necessary for transcription of an associateddownstream sequence. An expression vector is a plasmid containing anexpression construct. If appropriate and desired for the associatedsequence, the term expression also encompasses translation (proteinsynthesis) of the transcribed RNA. The particular sequences contained inthe expression vector include a promoter, enhancer, termination signal,transcriptional block and the like. To prevent transcriptionalinterference from multiple transgenes, a transcriptional block can beplaced between appropriate genes on a plant transformation plasmid. Apromoter is a DNA region that includes sequences sufficient to causetranscription of an associated (downstream) sequence. The promoter maybe regulated, i.e., not constitutively acting to cause transcription ofthe associated sequence. If inducible, there are sequences presenttherein that mediate regulation of expression so that the associatedsequence is transcribed only when an inducer molecule is present. In thepresent context, the inducer molecule is analogous to the signaltransmitted by an input circuit.

The term “derived from” includes genes, nucleic acids, and proteins whenthey include fragments or elements assembled in such a way that theyproduce a functional unit. The fragments or elements can be assembledfrom multiple organisms provided that they retain evolutionarilyconserved function. Elements or domains could be assembled from variousorganisms and/or synthesized partially or entirely, provided that theyretain evolutionarily conserved function, elements or domains. In somecases the derivation could include changes so that the codons areoptimized for expression in a particular organism.

“Expression control sequences” are DNA sequences involved in any way inthe control of transcription or translation. Suitable expression controlsequences and methods of making and using them are well-known in theart. The expression control sequences must include a promoter. Thepromoter may be any DNA sequence that shows transcriptional activity inthe chosen organism, plant cells, plant parts, or plants. The promotermay be inducible or constitutive. It may be naturally-occurring, may becomposed of portions of various naturally-occurring promoters, or may bepartially or totally synthetic. Also, the location of the promoterrelative to the transcription start may be optimized. Many suitablepromoters for use in plants are well-known in the art as are nucleotidesequences that enhance expression of an associated expressible sequence.

The term “RNA interfering molecule” includes, but is not limited to,diRNA, siRNA miRNA, or an antisense RNA to inhibit synthesis of arelated coding sequence. It is part of a mechanism for RNA-guidedregulation of gene expression in which double-stranded ribonucleic acid(RNA) inhibits the expression of genes with complementary nucleotidesequences.

The DNA constructs of the present disclosure can be used to transformany type of cell, plant or plant cell. A genetic marker can be used forselecting transformed cells (“a selection marker”). Selection markerstypically allow transformed cells to be recovered by negative selection(i.e., inhibiting growth of cells that do not contain the selectionmarker) or by screening for a product encoded by the selection marker.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples, which followrepresent techniques discovered by the inventors to function well in thepractice of the present disclosure, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments, which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the present disclosure.

Example 1: Using the N-Terminal End of the Signal Dependent HistidineKinase Activation Region to Engineer Novel Receptor Histidine KinaseFusions

Most of the reported functional chimeric receptor/HK fusions utilize acommon signaling domain, the HAMP domain, which is found in chemotacticreceptors and some HKs. The first reported chimeric receptor/HK fusionwas made by fusing a chemotactic receptor Tar to the HK EnvZ at theshared HAMP domain. However, there are a large number of receptors andHKs that lack a HAMP domain. PhoR lacks a HAMP domain which madedetermining a functional fusion point (functional=off in the absence ofligand and on in presence of the ligand) with previous knowledge andtechnology difficult. Previous fusions of Trg to PhoR showed a highbasal activity (kinase on) in the absence of signaling.

A computational learning algorithm can be used to show a proteinstructural feature, a coiled-coil like element, in the dimerization andhistidine phosphorylation (DHP) domain of HKs (Singh, et al., Proc.Natl. Acad. Sci. USA 95:2738-2743). As diagramed in FIG. 3 ,coiled-coils result from protein-protein interactions between 2 or morealpha helices somewhat resembling the coiled strands in a springs. Thereare seven amino acids per coiled coil alpha helix and the amino acidsare designated A-G. The interfaces between helices of coiled coils arecharacterized by interactions of the residues located at the A positionof one helix with the D position of a second helix. The interface of thetwo helices typically has hydrophobic residues at the A and D positions.When the alpha helices interact to form a coiled coil, residues from onehelix occupy the spaces between corresponding residues from the secondhelix, producing a “knobs in holes” packing.

The inventors reasoned that because this coiled-coil like element was acommon motif in histidine kinases it would be possible to make fusionsutilizing this element. In addition to strict fusions between Trg andPhoR, coiled coil elements were also incorporated from a functionalfusion between Trg and EnvZ (Trz) with the idea that it may containstructural elements that allow proper HAMP signaling activation of an HKwhereas PhoR, which lacks a HAMP domain, may not be able to be properlyactivated in the absence of these elements. The sequences of PhoR andTrz fusion were submitted to the HK coiled coil prediction program(groups.csail.mit.edu/cb/learncoil/cgi-bin/learncoil.cgi) (FIG. 4 ).Fusions incorporating the coiled-coil helices from Trz were designed aswell as a fusion using the PhoR coiled-coil and the equivalent region ofTrg which lacks the HK coiled coil (FIG. 5 ). These fusions were testedin E. coli. An expression plasmid was constructed in the plasmidpACYC177 with a LacI promoter driving RBP and a LacI promoter driving anoperon consisting of the PhoB response regulator and the chimericreceptor:PhoR fusion to be tested. The operon was modeled after thenaturally occurring PhoB/PhoR operon (FIG. 6 ). The expressionconstructs were transformed into the E. coli cell line BW23423, whichhas a PhoB responsive promoter driving a β-galactosidase reporter gene.The functionality of the fusions was tested by a split plate assay withX-Gal present to monitor (3-galactosidase activity. One side of thesplit plate contained medium with maltose as a control; the other sideof the split plate had medium containing ribose, the ligand being usedto test functionality. A functional fusion should show no signaling onmaltose (white colonies) whereas colonies growing on ribose should beblue. The blue color indicates the ligand activates the HK receptorfusion, which then transfers a phosphoryl group to PhoB leading to theactivation of the β-galactosidase reporter gene. Three of the coiledcoil based fusions showed high basal activity, but the fusionincorporating a coiled coil heptad of EnvZ (heptad repeat 3) wasfunctional.

Functional analysis within the 7 amino acids of the EnvZ coiled-coilelement revealed the key point of the fusion wasn't the first amino acidof the seven amino acids of the EnvZ coiled coil region (residue A) butthe 4^(th) amino acid (residue D), a valine (FIG. 7 ). Because theground state of a histidine kinase is such that the kinase is active,“kinase on” and the predominate signaling phenotype seen innon-functional fusions is “kinase on,” the inventors reasoned that thevaline at the D position is essential in maintaining the “kinase off”state in the absence of ligand dependent signaling. Fusions where the Dposition valine is replaced by alanine, glycine (the two hydrophobicamino acids smaller than valine) or a methionine (the D position residueof PhoR) results in constitutive activation (FIG. 8 ). E. coli has 10HKs, including EnvZ, with HAMP domains adjacent to the HK coiled coil(FIG. 8 ). EnvZ is the only HK with a valine in the A/D position. Thereare 4 other amino acids, glutamate, isoleucine, leucine and threoninethat occupy the A/D position in the 9 other E. coli HKs with a HAMP/HKcoiled coil A/D overlap. Fusions testing whether any of these aminoacids can functionally substitute for the valine were produced. Allfusions resulted in the high basal phenotype or “kinase on” suggestingthat the valine at the D position is essential in creating functionalreceptor/PhoR fusions. The A/D position represents the point at whichthe signal from the receptor acts to initiate histidine kinase activity.This position defines the N-terminal end of the signal dependenthistidine kinase activation region. The functional TrzPhoR fusionrepresents an improvement in the synthetic signaling system that enablesdetector plants allowing for a cleaner signaling system. In addition,the TrzPhoR fusion has been introduced into the plant detector systemand it was shown to be highly functional (FIG. 9 ).

To test the utility of the A/D position as a fusion point forengineering novel receptor/histidine kinase fusions, a fusion betweenthe RpfC receptor and PhoR was made. RpfC is a sensor HK involved inquorum sensing found in many species of Xylella and Xanthomonas that arebacterial pathogens of plants (Chatterjee, et al., Proc. Natl. Acad.Sci. USA 105:2670-2675, 2008). RpfC senses Diffusible Signaling Factor(DSF) mediating the control of virulence factor synthesis, and hencestart the virulence response. RpfC does not have a HAMP domain only acytoplasmic linker between the final transmembrane helix and the DHpdomain. RpfC has a predicted HK coiled coil in the same register as theone predicted for PhoR. The RpfC DSF sensor and cytoplasmic linker wasfused to PhoR at the A/D position. Both the D position of wild-type PhoRand the TrzPhoR fusion D position were used. The RpfCPhoR D positionfusion containing a methionine at the D position had a high basalphenotype. The RpfCTrzPhoR D position fusion containing a valine at theD position showed inducible signaling in the presence of DSF extract(FIG. 10 ). This result showed the efficacy of using the A/D position asa fusion point for receptor/histidine kinase fusions. These results alsoshowed how important the amino acid occupying the A/D position is inobtaining a functional fusion. In addition, when the RpfCTrzPhoR Dposition fusion was expressed in transgenic Arabidopsis plants, thepresence of DSF was detected using a luciferase reporter gene readout(FIG. 11A and FIG. 11B). An additional test of the D position fusionpoint was implemented by fusing the PhoQ receptor to PhoR. PhoQ is aMg²⁺ responsive receptor histidine kinase that mediates adaptation toMg²⁺ limiting environments (Groisman, J. Bacteriol. 183:1835-1842,2001). PhoQ also lacks a cytoplasmic HAMP domain. FIG. 12 demonstratesPhoQTrzPhoR showing an increase in Mg²⁺ signaling (above that seen fromthe Mg²⁺ already present in the media) with exposure to additional Mg²⁺.

Example 2: Using the C-Terminal End of the Signal Dependent HistidineKinase Activation Region to Engineer Inducible Kinase Activity inNon-Functional Receptor/Histidine Kinase Fusions

An engineered an EnvZ variant that interacts with PhoB was previouslyengineered by substituting twenty-eight amino acids from thehelix-loop-helix region of the PhoR DHp domain into the correspondingpositions of the EnvZ DHp domain. This version of EnvZ, called Chim3,was able to phosphorylate PhoB in an in vitro assay (Skerker, et al.,Cell 133:1043-1054, 2008). The changes in EnvZ that allowed it tointeract with PhoB were incorporated into the Trg receptor EnvZ fusionTrz (Baumgartner, et al., J. Bacteriol. 176:1157-1163, 1994). Thischimeric version of Trz was named TrzChim3. When TrzChim3 was testedin-vivo for the ability to signal through PhoB, high basal activity wasfound and no evidence of induction (FIG. 13A). This is termed as akinase “locked-ON” phenotype. PhoR and EnvZ differ in three amino acidsthat are located directly upstream from a conserved arginine found inthe DHp domain of both PhoR and EnvZ. The conserved arginine is in aregion proposed to be involved in the interaction between the CA domainand the DHp domain in the kinase OFF/phosphatase ON state. The inventorsreasoned that this area was important in activating histidine kinaseactivity and that this region of TrzChim3, which evolved to control EnvZkinase activation, was unable to control the activity of the chimericEnvZ/PhoR. In EnvZ the three amino acids are alanine, aspartate andaspartate (ADD) (residues 275-277, FIG. 14 ), while in PhoR the aminoacids are glutamate, glycine and alanine (EGA) (see PhoR alignment withTrz, FIG. 14 ). A version of TrzChim3 was constructed with EGAsubstituting for ADD. This substitution allowed for the determination ofwhether the three residues have a function in controlling the CA/DHpinteraction involved in the kinase off/phosphatase on state. Replacingthe three putative CA interaction region EnvZ amino acids with those ofPhoR did not restore ligand inducible function (FIG. 13B). However, thesubstitution did result in a change from a kinase “locked ON” phenotypeto a kinase “locked OFF” phenotype, suggesting that this region couldindeed be involved in the signal inducible activation of kinaseactivity.

The inventors reasoned that the chimeric nature of the DHphelix-loop-helix region in TrzChim3 results in a structure that does notcorrectly position the CA domain in the same way that the native EnvZ orPhoR CA domains are positioned. Further alterations to the putative CAinteraction region were tested to determine if these could provide afunctional HK. All three amino acid positions were randomly mutagenizedand screened for functionality by plating on media with the riboseligand and X-gal. Blue colonies were selected and re-tested for riboseinducibility on the split plates. Two mutants were isolated, TrzChim3-8and TrzChim3-10, that showed ligand inducible signaling with the splitplate assay (FIG. 15 ). The mutants were sequenced and it was found thatthe putative CA domain interaction region amino acids were mutated fromEGA to RGV in TrzChim3-8 and EGA to AGG in TrzChim3-10. FIG. 16 showsquantification of the kinase function for TrzChim3, TrzChim3-8 andTrzChim3-10. The ability to restore in vivo inducibility to TrzChim3suggests that the original TrzChim3 kinase “locked-ON” phenotype is dueto a perturbation of the CA/DHp domain interaction. This experimentallowed for the identification and manipulation of the C-terminal end ofthe signal dependent histidine kinase activation region.

Example 3—Development of a Maltose Inducible Tar HK Fusion

In E. coli, the chemotactic receptor Tar mediates chemotaxis towards theamino acid aspartate and the disaccharide sugar maltose via aninteraction between Tar and Maltose Binding Protein (MBP). When Tar isfused to the histidine kinase EnvZ to form Taz, the fusion retains theability to be induced by aspartate, however it no longer shows aresponse to maltose. Trg was replaced with Tar in either the TrzPhoRfusion to form TazPhoR or in TrzChim3-8 to form Tac8. When TazPhoR orTac8 was tested with maltose no maltose inducibility was observed.However, aspartate inducibility of both fusions was observed. In E.coli, aspartate mediates a stronger chemotactic response than maltose.The inventors reasoned that the transmembrane signal mediated by Tarwhen it binds aspartate is strong enough to activate histidine kinaseactivity in a Tar/HK fusion but the transmembrane signal mediated by Tarwhen it interacts with MBP is not strong enough to activate histidinekinase activity. Based on the ability to manipulate histidine kinaseactivity in TrzChim3, the inventors reasoned that targeting the samethree amino acids in TazPhoR or Tac8 may results in variants whosekinase activation threshold is lowered enough to be activated bymaltose. Amino acids 265, 266 and 267 in both Tac8 and TazPhoR weremutagenized by site directed mutagenesis. The site directed mutagenesislibraries were screened using fluorescent activated cell sorting (FACS).Initial sorting for Tac and TazPhoR functionality showed populations ofcells with significant maltose ligand induction. From this screen twoTazPhoR variants (TazPhoR 61 and 86; FIG. 17A) and one Tac8 variant(Tac40; FIG. 17B) were selected for further testing and development.This is the first reported example of MBP/maltose signaling via a TarHKfusion.

Example 4—Using TazPhoR61 and a Maltose Binding Protein with aComputationally Designed Binding Pocket to Detect a Fentanyl Ligand

The ability to computationally design proteins partially or entirely,including the ligand binding pockets of several Periplasmic BindingProteins, e.g., Maltose Binding Protein, Ribose Binding Protein, andGlucose Binding Protein, offers powerful means to produce new types ofprotein sensors and enable them in plants to serve as plant sentinels.Because previous TarHK fusions (Tar/EnvZ and Tar/PhoR) were unresponsiveto maltose signaling through MBP it was not possible to test whetherMBPs with computationally redesigned binding pockets could use HKsignaling to report the presence of a ligand of interest. Using an MBPredesigned to bind a Fentanyl ligand, the inventors tested whether themaltose inducible TazPhoR61 could be used to detect the presence ofFentanyl in the environment. Two genetic circuits were used in thisexperiment. A Fentanyl Detection circuit consisting of the redesignedMaltose Binding Protein (MBP 6.1-5), TazPhoR61 and PhoB wasco-transformed with a PhoB Signal Amplifying circuit consisting of aPhoB responsive promoter driving a Tal transcription factor engineeredto bind Gal4 binding sites, a Gal4 responsive promoter driving aGal4VP64 transcription factor (Gal4VP64 can bind its own promoterresulting in a positive feedback) and a Gal4 responsive promoter drivinga luciferase reporter gene. Transformed plants were exposed to 500 μMFentanyl and responded by inducing expression of luciferase (FIG. 18 ).The system described above is diagrammed in FIG. 19 with thequantitative controllers (positive feedback system) shown in FIG. 20 .

The term “about” is used herein to indicate that a value includes thestandard deviation of error for the device or method being employed todetermine the value. The use of the term “or” in the claims is used tomean “and/or” unless explicitly indicated to refer to alternatives onlyor the alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When not used in conjunction closed wording in the claims orspecifically noted otherwise, the words “a” and “an” denote “one ormore.”

The terms “comprise,” “have,” and “include” are open-ended linkingverbs. Any forms or tenses of one or more of these verbs, such as“comprises,” “comprising,” “has,” “having,” “includes,” and “including,”are also open-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps. Similarly, any cellthat “comprises,” “has” or “includes” one or more traits is not limitedto possessing only those one or more traits and covers other unlistedtraits.

While the disclosure has been described in connection with specificembodiments thereof, it will be understood that the present disclosureis capable of further modifications by one of skill in the art. It is tobe understood that, unless otherwise indicated, the present disclosureis not limited to particular materials, reagents, reaction materials,manufacturing processes, or the like, as such can vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting. It is also possible in the present disclosure that steps canbe executed in different sequence where this is logically possible. Thepresent disclosure is therefore intended to encompass any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein before set forth.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the method described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

All publications, patents, and patent publications cited areincorporated by reference herein in their entirety for all purposes.

What is claimed is:
 1. A fusion protein comprising a chemotacticreceptor protein, a receptor involved in quorum sensing, or a receptorfrom a receptor histidine kinase operably linked at the A/D position toa histidine kinase protein, wherein the fusion protein comprises akinase activation region.
 2. The fusion protein of claim 1, wherein thechemotactic receptor protein is Trg, Tar, Tap or Tsr.
 3. The fusionprotein of claim 1, wherein the receptor involved in quorum sensing isthe Xylella DSF receptor RpfC or the LuxPQ receptor LuxP.
 4. The fusionprotein of claim 1, wherein the histidine kinase protein is PhoR orEnvZ.
 5. The fusion protein of claim 1, wherein the histidine kinaseprotein is a EnvZ/PhoR chimera.
 6. The fusion protein of claim 1,wherein the kinase activation region of the fusion protein has beenengineered to restore inducible kinase activity or engineered to allowthe interaction of maltose-bound maltose binding protein with thereceptor to functionally activate kinase activity.
 7. The fusion proteinof claim 1, wherein the histidine kinase protein is activated when thechemotactic receptor protein or the receptor involved in quorum sensingbinds to a sensor protein bound to a target substance.
 8. The fusionprotein of claim 7, wherein the target substance is a chemical agent, aheavy metal, a poison, a pollutant, a toxin, an herbicide, a polycyclicaromatic hydrocarbon, a benzene, a toluene, a xylene, a halogenatedhydrocarbon, a steroid or other hormone, an explosive, or a degradationproduct of one of the foregoing compounds.
 9. The fusion protein ofclaim 1, further comprising a plasma membrane targeting signal sequenceoperably linked to an N-terminus of the chemotactic receptor protein orreceptor involved in quorum sensing.
 10. A DNA construct comprising anucleic acid segment that encodes the fusion protein of claim
 1. 11. TheDNA construct of claim 10, wherein the nucleic acid segment is operablylinked to a promoter.
 12. A transgenic plant comprising: a) a first DNAconstruct comprising a first plant operable promoter operably linked toa nucleic acid segment encoding a sensor protein, said proteincomprising a secretory sequence for directing the protein to theextracellular space of a plant cell and a binding region specific for atarget substance of interest, wherein said protein undergoes aconformational change when the target substance is bound; b) a secondDNA construct comprising a second plant operable promoter operablylinked to a nucleic acid segment encoding a protein that comprises thefollowing domains: a plasma membrane targeting signal sequence, anextracellular domain for binding the sensor protein, a transmembranedomain and a histidine kinase domain for phosphorylating a protein withnuclear shuttling or transcriptional activating functions, wherein thehistidine kinase is activated when the sensor protein binds to theextracellular domain; and c) a third DNA construct comprising a thirdplant operable promoter operably linked to a nucleic acid segmentencoding a detectable marker or a response gene, wherein the third plantoperative promoter is responsive to the transcriptional activatorprotein, and wherein the detectable marker is expressed when theexternal target substance of interest is bound to the sensor protein.13. The transgenic plant of claim 12, wherein the transmembrane domainand the histidine kinase domain of the second DNA construct are derivedfrom one or more bacterial genes, and the membrane targeting signalsequence of the second DNA construct is derived from a plant gene. 14.The transgenic plant of claim 12, wherein the detectable marker of thethird DNA construct is a chlorophyll degradation enzyme or a functionalfragment thereof.
 15. A method for detecting an external substance ofinterest, the method comprising: a) exposing the transgenic plant ofclaim 12 to an external substance of interest; and b) detecting a changeresulting from expression of the detectable marker.